Abuse-resistant amphetamine prodrugs

ABSTRACT

The invention describes compounds, compositions, and methods of using the same comprising a chemical moiety covalently attached to amphetamine. These compounds and compositions are useful for reducing or preventing abuse and overdose of amphetamine. These compounds and compositions find particular use in providing an abuse-resistant alternative treatment for certain disorders, such as attention deficit hyperactivity disorder (ADHD), ADD, narcolepsy, and obesity. Oral bioavailability of amphetamine is maintained at therapeutically useful doses. At higher doses bioavailability is substantially reduced, thereby providing a method of reducing oral abuse liability. Further, compounds and compositions of the invention decrease the bioavailability of amphetamine by parenteral routes, such as intravenous or intranasal administration, further limiting their abuse liability.

CROSS REFERENCE RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/400,304, filed Apr. 10, 2006, which in turn, (a) claims benefit under35 U.S.C. 119(e) to U.S. Provisional Application Nos. 60/669,385 filedApr. 8, 2005, 60/669,386 filed Apr. 8, 2005, 60/681,170 filed May 16,2005, 60/756,548 filed Jan. 6, 2006, and 60/759,958 filed Jan. 19, 2006;(b) is a continuation-in-part of U.S. application Ser. No. 10/857,619filed Jun. 1, 2004, now U.S. Pat. No. 7,223,735, which claims thebenefit of under 35 U.S.C. 119(e) to U.S. Provisional Application Nos.60/473,929 filed May 29, 2003 and 60/567,801 filed May 5, 2004; and (c)is a continuation-in-part of U.S. application Ser. No. 10/858,526 filedJun. 1, 2004, now U.S. Pat. No. 7,105,486, which, in turn, is acontinuation-in-part of international application PCT/US03/05525 filedFeb. 24, 2003, which claims priority to U.S. Provisional ApplicationNos. 60/358,368 filed Feb. 22, 2002 and 60/362,082 filed Mar. 7, 2002;application Ser. No. 10/858,526 also claims benefit under 35 U.S.C.119(e) to U.S. Provisional Application Nos. 60/473,929 filed May 29,2003 and 60/567,801 filed May 5, 2004. All of the above-identifiedapplications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to amphetamine compounds, more particularly toamphetamine prodrugs comprising amphetamine covalently bound to achemical moiety. The invention also relates to pharmaceuticalcompositions comprising the amphetamine compounds, and to methods ofmanufacturing, delivering, and using the amphetamine compounds.

BACKGROUND OF THE INVENTION

Amphetamines stimulate the central nervous system (CNS) and have beenused medicinally to treat various disorders including attention deficithyperactivity disorder (ADHD), obesity, and narcolepsy. In children withADHD, potent CNS stimulants have been used for several decades as a drugtreatment given either alone or as an adjunct to behavioral therapy.While methylphenidate (Ritalin®) has been the most frequently prescribedstimulant, the prototype of the class, amphetamine (alpha-methylphenethylamine) has been used all along and increasingly so in recentyears. (Bradley C, Bowen M, “Amphetamine (benzedrine) therapy ofchildren's behavior disorders.” American Journal of Orthopsychiatry 11:92-103 (1941).

Because of their stimulating effects, amphetamines, includingamphetamine derivatives and analogs, are subject to abuse. A user canbecome dependent over time on these drugs and their physical andpsychological effects, even when the drugs are used for legitimatetherapeutic purposes. Legitimate amphetamine users that develop drugtolerances are especially susceptible to becoming accidental addicts asthey increase dosing in order to counteract their increased tolerance ofthe prescribed drugs. Additionally, it is possible for individuals toinappropriately self-administer higher than prescribed quantities of thedrug or to alter either the product or the route of administration(e.g., inhalation (snorting), injection, and smoking), potentiallyresulting in immediate release of the active drug in quantities largerthan prescribed. When taken at higher than prescribed doses,amphetamines can cause temporary feelings of exhilaration and increasedenergy and mental alertness.

Recent developments in the abuse of prescription drug productsincreasingly raise concerns about the abuse of amphetamine prescribedfor ADHD. The National Survey on Drug Use and Health (NSDUH), estimatesthat in 2003, 1.2 million Americans aged 12 and older abused stimulants,such as amphetamines. The high abuse potential has earned amphetaminesSchedule II status according to the Controlled Substances Act (CSA).Schedule II classification is reserved for those drugs that haveaccepted medical use but have the highest potential for abuse.

Sustained release formulations of amphetamines, e.g., Adderall XR®, havean increased abuse liability relative to the single dose tablets becauseeach tablet of the sustained release formulation contains a higherconcentration of amphetamine. It may be possible for substance abusersto obtain a high dose of amphetamine with rapid onset by crushing thetablets into powder and snorting it or by dissolving the powder in waterand injecting it. Sustained release formulations may also provide unevenrelease.

Additional information about amphetamines and amphetamine abuse can befound in U.S. Publication No. 2005/0054561 (U.S. Ser. No. 10/858,526).

The need exists for additional amphetamine compounds, especially abuseresistant amphetamine compounds. Further, the need exists foramphetamine pharmaceutical compositions that provide sustained releaseand sustained therapeutic effect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Synthesis of peptide amphetamine conjugates.

FIG. 2. Synthesis of lysine amphetamine dimesylate.

FIG. 3. Synthesis of lysine amphetamine HCl.

FIG. 4. Synthesis of serine amphetamine conjugate.

FIG. 5. Synthesis of phenylalanine amphetamine conjugate.

FIG. 6. Synthesis of triglycine amphetamine conjugate.

FIG. 7. Plasma concentrations of d-amphetamine from individual ratsorally administered d-amphetamine or L-lysine-d-amphetaminehydrochloride.

The following Figures (FIG. 8-FIG. 16) depict results obtained fromstudies of oral administration of d-amphetamine sulfate orL-lysine-d-amphetamine dimesylate to rats (ELISA analysis):

FIG. 8. Plasma concentrations of d-amphetamine (at dose 1.5 mg/kgd-amphetamine base).

FIG. 9. Plasma concentrations of d-amphetamine (at dose 3 mg/kgd-amphetamine base).

FIG. 10. Plasma concentrations of d-amphetamine (at dose 6 mg/kgd-amphetamine base).

FIG. 11. Plasma concentrations of d-amphetamine (at dose 12 mg/kgd-amphetamine base).

FIG. 12. Plasma concentrations of d-amphetamine (at dose 30 mg/kgd-amphetamine base).

FIG. 13. Plasma concentrations of d-amphetamine (at dose 60 mg/kgd-amphetamine base).

FIG. 14. Percent bioavailability (AUC and C_(max)) ofL-lysine-d-amphetamine dimesylate compared to d-amphetamine sulfate atdoses 1.5, 3, 6, 12, 30, and 60 mg/kg d-amphetamine base.

FIG. 15. Plasma concentrations of d-amphetamine at 30-minutes post-dosefor escalating doses of d-amphetamine base.

FIG. 16. Plasma concentrations of d-amphetamine (at dose 60 mg/kgd-amphetamine base).

FIG. 17. Plasma concentrations of d-amphetamine following intranasaladministration of L-lysine-d-amphetamine hydrochloride or d-amphetaminesulfate (at dose 3 mg/kg d-amphetamine base) to rats (ELISA analysis).

FIG. 18. Plasma concentrations of d-amphetamine following intranasaladministration of L-lysine-d-amphetamine dimesylate or d-amphetaminesulfate (at dose 3 mg/kg d-amphetamine base) to rats (ELISA analysis).

FIG. 19. Plasma concentrations of d-amphetamine following bolusintravenous administration of L-lysine-d-amphetamine dimesylate ord-amphetamine sulfate (at dose 1.5 mg/kg d-amphetamine base) to rats(ELISA analysis).

FIG. 20. Plasma concentrations of d-amphetamine levels following oraladministration of Dexedrine Spansule® capsules, crushed DexedrineSpansule® capsules, or L-lysine-d-amphetamine dimesylate (at dose 3mg/kg d-amphetamine base) to rats (ELISA analysis).

The following Figures (FIG. 21-FIG. 30) depict results obtained fromstudies of oral administration of d-amphetamine sulfate orL-lysine-d-amphetamine dimesylate to rats (LC/MS/MS analysis):

FIG. 21A and FIG. 21B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 21A) and in nM (FIG. 21B) (at dose 1.5 mg/kg d-amphetamine base).

FIG. 22A and FIG. 22B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 22A) and in nM (FIG. 22B) (at dose 3 mg/kg d-amphetamine base).

FIG. 23A and FIG. 23B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 23A) and in nM (FIG. 23B) (at dose 6 mg/kg d-amphetamine base).

FIG. 24A and FIG. 24B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 24A) and in nM (FIG. 24B) (at dose 12 mg/kg d-amphetamine base).

FIG. 25A and FIG. 25B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 25A) and in nM (FIG. 25B) (at dose 60 mg/kg d-amphetamine base).

FIG. 26. Comparative bioavailability (C_(max)) of L-lysine-d-amphetamineand d-amphetamine in proportion to escalating human equivalent doses.

FIG. 27. Comparative bioavailability (AUC_(inf)) ofL-lysine-d-amphetamine and d-amphetamine in proportion to escalatingdoses of d-amphetamine base.

FIG. 28. Comparative bioavailability (AUC_(inf)) ofL-lysine-d-amphetamine and d-amphetamine in proportion to escalatinghuman equivalent doses.

FIG. 29. Comparative bioavailability (C_(max)) of intactL-lysine-d-amphetamine in proportion to escalating human equivalentdoses.

FIG. 30. Comparative bioavailability (AUC_(inf)) of intactL-lysine-d-amphetamine in proportion to escalating human equivalentdoses.

FIG. 31. Plasma concentrations of d-amphetamine following intranasaladministration of L-lysine-d-amphetamine dimesylate or d-amphetaminesulfate (at dose 3 mg/kg d-amphetamine base) to rats (LC/MS/MSanalysis).

FIG. 32A and FIG. 32B. Plasma concentrations of d-amphetamine andL-lysine-d-amphetamine in ng/mL (FIG. 32A) and in nM (FIG. 32B),following intranasal administration of L-lysine-d-amphetamine dimesylateor d-amphetamine sulfate (at dose 3 mg/kg d-amphetamine base) to rats(LC/MS/MS analysis).

FIG. 33. Plasma concentrations of d-amphetamine following bolusintravenous administration of L-lysine-d-amphetamine dimesylate ord-amphetamine sulfate (at dose 1.5 mg/kg d-amphetamine base) to rats(LC/MS/MS analysis).

FIG. 34A and FIG. 34B. Plasma concentrations of d-amphetamine in ng/mL(FIG. 34A) and in nM (FIG. 34B), following intravenous administration ofL-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.5mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

The following Figures (FIG. 35-FIG. 40) depict results obtained fromstudies of oral and intravenous administration (at dose 1 mg/kgd-amphetamine base) of d-amphetamine sulfate or L-lysine-d-amphetaminedimesylate to conscious male beagle dogs (LC/MS/MS analysis):

FIG. 35. Mean plasma concentration time profile ofL-lysine-d-amphetamine following intravenous or oral administration ofL-lysine-d-amphetamine (n=3).

FIG. 36. Plasma concentration time profile of d-amphetamine followingintravenous or oral administration of L-lysine-d-amphetamine (n=3).

FIG. 37A and FIG. 37B. Mean plasma concentration time profile ofL-lysine-d-amphetamine and d-amphetamine levels in ng/ml (FIG. 37A) andin nM (FIG. 37B), following intravenous administration ofL-lysine-d-amphetamine (n=3).

FIG. 38A and FIG. 38B. Mean plasma concentration time profile ofL-lysine-d-amphetamine and d-amphetamine levels in ng/ml (FIG. 38A) andin nM (FIG. 38B), following oral administration ofL-lysine-d-amphetamine (n=3).

FIG. 39A and FIG. 39B. Individual plasma concentration time profile ofL-lysine-d-amphetamine following intravenous administration (FIG. 39A)or oral administration (FIG. 39B) of L-lysine-d-amphetamine.

FIG. 40A and FIG. 40B. Individual plasma concentration time profile ofd-amphetamine following intravenous administration (FIG. 40A) or oraladministration (FIG. 40B) of L-lysine-d-amphetamine.

FIG. 41. Plasma concentrations of d-amphetamine following oraladministration of L-lysine-d-amphetamine dimesylate or d-amphetaminesulfate (at dose 1.8 mg/kg d-amphetamine base) to male dogs.

FIG. 42. Plasma concentrations of d-amphetamine following oraladministration of L-lysine-d-amphetamine dimesylate or d-amphetaminesulfate (at dose 1.8 mg/kg d-amphetamine base) to female dogs.

FIG. 43. Mean blood pressure following intravenous injection ofincreasing amounts of L-lysine-d-amphetamine dimesylate or d-amphetaminein male and female dogs.

FIG. 44. Left ventricular blood pressure following intravenous injectionof increasing amounts of L-lysine-d-amphetamine dimesylate ord-amphetamine in male and female dogs.

The following Figures (FIG. 45-FIG. 49) depict results obtained fromstudies of oral (at dose 6 mg/kg d-amphetamine base), intranasal (atdose 1 mg/kg d-amphetamine base), and intravenous administration (atdose 1 mg/kg d-amphetamine base) of d-amphetamine sulfate orL-lysine-d-amphetamine hydrochloride to rats:

FIG. 45. Locomotor activity of rats following oral administration (5hour time-course).

FIG. 46. Locomotor activity of rats following oral administration (12hour time-course).

FIG. 47. Locomotor activity of rats following intranasal administration(1 hour time-course).

FIG. 48. Locomotor activity of rats following intranasal administration(with carboxymethylcellulose) (2 hour time-course).

FIG. 49. Locomotor activity of rats following intravenous administration(3 hour time-course).

The following Figures (FIG. 50-FIG. 58) depict results obtained fromstudies of oral, intranasal, and intravenous administration ofd-amphetamine or amphetamine conjugate hydrochloride salts to rats(ELISA analysis):

FIG. 50. Intranasal bioavailability of abuse-resistant amphetamine aminoacid, di-, and tri-peptide conjugates.

FIG. 51. Oral bioavailability of abuse-resistant amphetamine amino acid,di-, and tri-peptide conjugates.

FIG. 52. Intravenous bioavailability of an abuse-resistant amphetaminetri-peptide conjugate.

FIG. 53. Intranasal bioavailability of an abuse-resistant amphetamineamino acid conjugate.

FIG. 54. Oral bioavailability of an abuse-resistant amphetamine aminoacid conjugate.

FIG. 55. Intravenous bioavailability of an abuse-resistant amphetamineamino acid conjugate.

FIG. 56. Intranasal bioavailability of an abuse-resistant amphetamineamino tri-peptide conjugate.

FIG. 57. Intranasal bioavailability of abuse-resistant amphetamine aminoacid-, and di-peptide conjugates.

FIG. 58. Intranasal bioavailability of an abuse-resistant amphetaminedi-peptide conjugate containing D- and L-amino acid isomers.

FIG. 59A and FIG. 59B. Plasma concentrations of d-amphetamine andL-lysine-d-amphetamine in ng/mL for the serum levels (FIG. 59A) and inng/g for brain tissue (FIG. 59B), following oral administration ofL-lysine-d-amphetamine hydrochloride or d-amphetamine sulfate (at dose 5mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

FIG. 60. Steady-state plasma d-amphetamine and L-lysine-d-amphetaminelevels obtained from clinical studies of oral administration ofL-lysine-d-amphetamine dimesylate 70 mg to humans (LC/MS/MS analysis).

The following Figures (FIG. 61-FIG. 70) depict results obtained fromclinical studies of oral administration of L-lysine-d-amphetaminedimesylate to humans (LC/MS/MS analysis):

FIG. 61A and FIG. 61B. Plasma d-amphetamine and L-lysine-d-amphetaminelevels (FIG. 61A, ng/mL; FIG. 61B, nM) over a 72 hour period followingoral administration of L-lysine-d-amphetamine (25 mgL-lysine-d-amphetamine dimesylate containing 7.37 mg d-amphetamine base)to humans.

FIG. 62A and FIG. 62B. Plasma d-amphetamine and L-lysine-d-amphetaminelevels (FIG. 62A, ng/mL; FIG. 62B, nM) over a 72 hour period followingoral administration of L-lysine-d-amphetamine (75 mgL-lysine-d-amphetamine dimesylate containing 22.1 mg d-amphetamine base)to humans.

FIG. 63A and FIG. 63B. Plasma d-amphetamine levels (FIG. 63A, 0-12hours; FIG. 63B, 0-72 hours) following oral administration ofL-lysine-d-amphetamine (75 mg L-lysine-d-amphetamine dimesylatecontaining 22.1 mg d-amphetamine base) or Adderall XR® (35 mg containing21.9 mg amphetamine base) to humans.

FIG. 64A and FIG. 64B. Plasma d-amphetamine levels (FIG. 64A, 0-12hours; FIG. 64B, 0-72 hours) following oral administration ofL-lysine-d-amphetamine (75 mg L-lysine-d-amphetamine dimesylatecontaining 22.1 mg d-amphetamine base) or Dexedrine Spansule® (30 mgcontaining 22.1 mg amphetamine base) to humans.

FIG. 65. Mean plasma concentration of d-amphetamine after oraladministration of single 30 mg, 50 mg, and 70 mg doses ofL-lysine-d-amphetamine dimesylate under fasted conditions to pediatricpatients with ADHD.

FIG. 66. Relationship between the dose-normalized AUC of d-amphetamineand gender after oral administration of L-lysine-d-amphetaminedimesylate capsules once daily to healthy adult volunteers and childrenwith ADHD.

FIG. 67. Relationship between the dose-normalized maximum plasmaconcentration of d-amphetamine and gender after oral administration ofL-lysine-d-amphetamine dimesylate capsules once daily to healthy adultvolunteers and children with ADHD.

FIG. 68. Relationship between the dose-normalized time to maximumconcentration of d-amphetamine and gender after oral administration ofL-lysine-d-amphetamine dimesylate capsules once daily to healthy adultvolunteers and children with ADHD.

FIG. 69. ADHD-RS at endpoint for pediatric clinical study.

FIG. 70. SKAMP score (efficacy) vs. time for pediatric clinical study.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides amphetamine prodrugs comprising amphetaminecovalently bound to a chemical moiety. The amphetamine prodrugs can alsobe characterized as conjugates in that they possess a covalentattachment. They may also be characterized as conditionallybioreversible derivatives (“CBDs”) in that the amphetamine prodrugpreferably remains inactive until oral administration releases theamphetamine from the chemical moiety.

In one embodiment, the invention provides an amphetamine prodrug ofFormula I:

A-X_(n)-Z_(m)  (I)

wherein A is an amphetamine;

each X is independently a chemical moiety;

each Z is independently a chemical moiety that acts as an adjuvant andis different from at least one X;

n is an increment from 1 to 50, preferably 1 to 10; and

m is an increment from 0 to 50, preferably 0.

When m is 0, the amphetamine prodrug is a compound of Formula (II):

A-X_(n)  (II)

wherein each X is independently a chemical moiety.

Formula (II) can also be written to designate the chemical moiety thatis physically attached to the amphetamine:

A-X₁—(X)_(n-1)  (III)

wherein A is an amphetamine; X₁ is a chemical moiety, preferably asingle amino acid; each X is independently a chemical moiety that is thesame as or different from X₁; and n is an increment from 1 to 50.

The amphetamine, A, can be any of the sympathomimetic phenethylaminederivatives which have central nervous system stimulant activity such asamphetamine, or any derivative, analog, or salt thereof. Exemplaryamphetamines include, but are not limited to, amphetamine,methamphetamine, methylphenidate, p-methoxyamphetamine,methylenedioxyamphetamine, 2,5-dimethoxy-4-methylamphetamine,2,4,5-trimethoxyamphetamine, and 3,4-methylenedioxymethamphetamine,N-ethylamphetamine, fenethylline, benzphetamine, and chlorphentermine aswell as the amphetamine compounds of Adderall®; actedron; actemin;adipan; akedron; allodene; alpha-methyl-(±)-benzeneethanamine;alpha-methylbenzeneethanamine; alpha-methylphenethylamine; amfetamine;amphate; anorexine; benzebar; benzedrine; benzyl methyl carbinamine;benzolone; beta-amino propylbenzene; beta-phenylisopropylamine;biphetamine; desoxynorephedrine; dietamine; DL-amphetamine; elastonon;fenopromin; finam; isoamyne; isomyn; mecodrin; monophos; mydrial;norephedrane; novydrine; obesin; obesine; obetrol; octedrine; oktedrin;phenamine; phenedrine; phenethylamine, alpha-methyl-; percomon;profamina; profetamine; propisamine; racephen; raphetamine; rhinalator,sympamine; simpatedrin; simpatina; sympatedrine; and weckamine.Preferred amphetamines include methamphetamine, methylphenidate, andamphetamine, with amphetamine being most preferred.

The amphetamine can have any stereogenic configuration, including bothdextro- and levo-isomers. The dextro-isomer, particularlydextroamphetamine, is preferred.

Preferably, the amphetamine is an amphetamine salt. Pharmaceuticallyacceptable salts, e.g., non-toxic, inorganic and organic acid additionsalts, are known in the art. Exemplary salts include, but are notlimited to, 2-hydroxyethanesulfonate, 2-naphthalenesulfonate,3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate, alginate,amsonate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate,bisulfate, bitartrate, borate, butyrate, calcium edetate, camphorate,camphorsulfonate, camsylate, carbonate, citrate, clavulariate,cyclopentanepropionate, digluconate, dodecylsulfate, edetate, edisylate,estolate, esylate, ethanesulfonate, finnarate, gluceptate,glucoheptanoate, gluconate, glutamate, glycerophosphate,glycollylarsanilate, hemisulfate, heptanoate, hexafluorophosphate,hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, laurylsulphonate, malate, maleate, mandelate,mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate,mucate, naphthylate, napsylate, nicotinate, nitrate, N-methylglucamineammonium salt, oleate, oxalate, palmitate, pamoate, pantothenate,pectinate, persulfate, phosphate, phosphateldiphosphate, picrate,pivalate, polygalacturonate, propionate, p-toluenesulfonate, saccharate,salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate,suramate, tannate, tartrate, teoclate, thiocyanate, tosylate,triethiodide, undecanoate, and valerate salts, and the like. (See Bergeet al. (1977) “Pharmaceutical Salts”, J Pharm. Sci. 66:1-19). Apreferred amphetamine salt is the mesylate salt (e.g., as inL-lysine-d-amphetamine dimesylate).

Particular salts may be less hygroscopic thereby facilitating handling.In a preferred embodiment, the amphetamine prodrug has a water content(Karl Fischer analysis) of about 0% to about 5%, about 0.1% to about 3%,about 0.25% to about 2%, or increments therein. When the amphetamineprodrug is formulated into a pharmaceutical composition, thepharmaceutical composition preferably has a water content of about 1% toabout 10%, about 1% to about 8%, about 2% to about 7%, or incrementstherein.

Throughout this application, the term “increment” is used to define anumerical value in varying degrees of precision, e.g., to the nearest10, 1, 0.1, 0.01, etc. The increment can be rounded to any measurabledegree of precision. For example, the range 1 to 100 or incrementstherein includes ranges such as 20 to 80, 5 to 50, 0.4 to 98, and 0.04to 98.05.

The amphetamine is bound to one or more chemical moieties, denominated Xand Z. A chemical moiety can be any moiety that decreases thepharmacological activity of amphetamine while bound to the chemicalmoiety as compared to unbound (free) amphetamine. The attached chemicalmoiety can be either naturally occurring or synthetic. Exemplarychemical moieties include, but are not limited to, peptides, includingsingle amino acids, dipeptides, tripeptides, oligopeptides, andpolypeptides; glycopeptides; carbohydrates; lipids; nucleosides; nucleicacids; and vitamins. Preferably, the chemical moiety is generallyrecognized as safe (“GRAS”).

“Carbohydrates” include sugars, starches, cellulose, and relatedcompounds, e.g., (CH₂O)_(n) wherein n is an integer larger than 2, andC_(n)(H₂O)_(n-1) wherein n is an integer larger than 5. The carbohydratecan be a monosaccharide, disaccharide, oligosaccharide, polysaccharide,or a derivative thereof (e.g., sulfo- or phospho-substituted). Exemplarycarbohydrates include, but are not limited to, fructose, glucose,lactose, maltose, sucrose, glyceraldehyde, dihydroxyacetone, erythrose,ribose, ribulose, xylulose, galactose, mannose, sedoheptulose,neuraminic acid, dextrin, and glycogen.

A “glycopeptide” is a carbohydrate linked to an oligopeptide. Similarly,the chemical moiety can also be a glycoprotein, glyco-amino-acid, orglycosyl-amino-acid. A “glycoprotein” is a carbohydrate (e.g., a glycan)covalently linked to a protein. A “glyco-amino-acid” is a carbohydrate(e.g., a saccharide) covalently linked to a single amino acid. A“glycosyl-amino-acid” is a carbohydrate (e.g., a saccharide) linkedthrough a glycosyl linkage (O—, N—, or S—) to an amino acid.

A “peptide” includes a single amino acid, a dipeptide, a tripeptide, anoligopeptide, a polypeptide, or a carrier peptide. An oligopeptideincludes from 2 to 70 amino acids.

Preferably, the chemical moiety is a peptide, more particularly a singleamino acid, a dipeptide, or a tripeptide. The peptide preferablycomprises fewer than 70 amino acids, fewer than 50 amino acids, fewerthan 10 amino acids, or fewer than 4 amino acids. When the chemicalmoiety is one or more amino acids, the amphetamine is preferably boundto lysine, serine, phenylalanine, or glycine. In another embodiment, theamphetamine is preferably bound to lysine, glutamic acid, or leucine. Inone embodiment, the amphetamine is bound to lysine and optionaladditional chemical moieties, e.g., additional amino acids. In apreferred embodiment, the amphetamine is bound to a single lysine aminoacid.

In one embodiment, the chemical moiety is from 1 to 12 amino acids,preferably 1 to 8 amino acids. In another embodiment, the number ofamino acids is 1, 2, 3, 4, 5, 6, or 7. In another embodiment, themolecular weight of the chemical moiety is below about 2,500 kD, morepreferably below about 1,000 kD, and most preferably below about 500 kD.

Each amino acid can be any one of the L- or D-enantiomers, preferablyL-enantiomers, of the naturally occurring amino acids: alanine (Ala orA), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp orD), cysteine (Cys or C), glycine (Gly or G), glutamic acid (Glu or E),glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I),leucine (Leu or L), lysine (Lys or K), methionine (Met or M), proline(Pro or P), phenylalanine (Phe or F), serine (Ser or S), tryptophan (Trpor W), threonine (Thr or T), tyrosine (Tyr or Y), and valine (Val or V).In a preferred embodiment, the peptide comprises only naturallyoccurring amino acids and/or only L-amino acids. Each amino acid can bean unnatural, non-standard, or synthetic amino acids, such asaminohexanoic acid, biphenylalanine, cyclohexylalanine,cyclohexylglycine, diethylglycine, dipropylglycine,2,3-diaminoproprionic acid, homophenylalanine, homoserine, homotyrosine,naphthylalanine, norleucine, ornithine, phenylalanine (4-fluoro),phenylalanine(2,3,4,5,6-pentafluoro), phenylalanine(4-nitro),phenylglycine, pipecolic acid, sarcosine,tetrahydroisoquinoline-3-carboxylic acid, and tert-leucine. Preferably,synthetic amino acids with alkyl side chains are selected from C₁-C₁₇alkyls, preferably C₁-C₆ alkyls. In one embodiment, the peptidecomprises one or more amino acid alcohols, e.g., serine and threonine.In another embodiment, the peptide comprises one or more N-methyl aminoacids, e.g., N-methyl aspartic acid.

In one embodiment, the peptides are utilized as base short chain aminoacid sequences and additional amino acids are added to the terminus orside chain. In another embodiment, the peptide may have an one or moreamino acid substitutions. Preferably, the substitute amino acid issimilar in structure, charge, or polarity to the replaced amino acid.For instance, isoleucine is similar to leucine, tyrosine is similar tophenylalanine, serine is similar to threonine, cysteine is similar tomethionine, alanine is similar to valine, lysine is similar to arginine,asparagine is similar to glutamine, aspartic acid is similar to glutamicacid, histidine is similar to proline, and glycine is similar totryptophan.

The peptide can comprise a homopolymer or heteropolymer of naturallyoccurring or synthetic amino acids. For example, the side chainattachment of amphetamine to the peptide can be a homopolymer orheteropolymer containing glutamic acid, aspartic acid, serine, lysine,cysteine, threonine, asparagine, arginine, tyrosine, or glutamine.

Exemplary peptides include Lys, Ser, Phe, Gly-Gly-Gly, Leu-Ser, Leu-Glu,homopolymers of Glu and Leu, and heteropolymers of (Glu)_(n)-Leu-Ser. Ina preferred embodiment, the peptide is Lys, Ser, Phe, or Gly-Gly-Gly.

In one embodiment, the chemical moiety has one or more free carboxyand/or amine terminal and/or side chain group other than the point ofattachment to the amphetamine. The chemical moiety can be in such a freestate, or an ester or salt thereof.

The chemical moiety can be covalently attached to the amphetamine eitherdirectly or indirectly through a linker. Covalent attachment maycomprise an ester or carbonate bond. The site of attachment typically isdetermined by the functional group(s) available on the amphetamine. Forexample, a peptide can be attached to an amphetamine via the N-terminus,C-terminus, or side chain of an amino acid. For additional methods ofattaching amphetamine to various exemplary chemical moieties, see U.S.application Ser. No. 10/156,527, PCT/US03/05524, and PCT/US03/05525,each of which is hereby incorporated by reference in its entirety.

The amphetamine prodrug compounds described above can be synthesized asdescribed in Example 1 and FIG. 1. Preferably, additional purificationand/or crystallization steps are not necessary to yield a highly pureproduct. In one embodiment, the purity of the amphetamine prodrug is atleast about 95%, more preferably at least about 96%, 97%, 98%, 98.5%,99%, 99.5%, 99.9%, or increments therein. For the synthesis ofL-lysine-d-amphetamine, known impurities include Lys-Lys-d-amphetamine,Lys(Lys)-d-amphetamine, d-amphetamine, Lys(Boc)-d-amphetamine,Boc-Lys-d-amphetamine, and Boc-Lys(Boc)-d-amphetamine. In oneembodiment, the presence of any single impurity is less than about 3%,more preferably less than about 2%, 1%, 0.5%, 0.25%, 0.15%, 0.1%, 0.05%,or increments therein.

In one embodiment, the amphetamine prodrug (a compound of one of theformulas described above) may exhibit one or more of the followingadvantages over free amphetamines. The amphetamine prodrug may preventoverdose by exhibiting a reduced pharmacological activity whenadministered at higher than therapeutic doses, e.g., higher than theprescribed dose. Yet when the amphetamine prodrug is administered attherapeutic doses, the amphetamine prodrug may retain similarpharmacological activity to that achieved by administering unboundamphetamine, e.g., Adderall XR®. Also, the amphetamine prodrug mayprevent abuse by exhibiting stability under conditions likely to beemployed by illicit chemists attempting to release the amphetamine. Theamphetamine prodrug may prevent abuse by exhibiting reducedbioavailability when it is administered via parenteral routes,particularly the intravenous (“shooting”), intranasal (“snorting”),and/or inhalation (“smoking”) routes that are often employed in illicituse. Thus, the amphetamine prodrug may reduce the euphoric effectassociated with amphetamine abuse. Thus, the amphetamine prodrug mayprevent and/or reduce the potential of abuse and/or overdose when theamphetamine prodrug is used in a manner inconsistent with themanufacturer's instructions, e.g., consuming the amphetamine prodrug ata higher than therapeutic dose or via a non-oral route ofadministration.

Use of phrases such as “decreased”, “reduced”, “diminished”, or“lowered” includes at least a 10% change in pharmacological activitywith greater percentage changes being preferred for reduction in abusepotential and overdose potential. For instance, the change may also begreater than 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%,or other increments greater than 10%.

Use of the phrase “similar pharmacological activity” means that twocompounds exhibit curves that have substantially the same AUC, C_(max),T_(max), C_(min), and/or t_(1/2) parameters, preferably within about 30%of each other, more preferably within about 25%, 20%, 10%, 5%, 2%, 1%,or other increments less than 30%.

Preferably, the amphetamine prodrug exhibits an unbound amphetamine oralbioavailability of at least about 60% AUC (area under the curve), morepreferably at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, orother increments greater than 60%. Preferably, the amphetamine prodrugexhibits an unbound amphetamine parenteral, e.g., intranasal,bioavailability of less than about 70% AUC, more preferably less thanabout 50%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or other incrementsless than 70%. For certain treatments, it is desirable that theamphetamine prodrug exhibits both the oral and parenteralbioavailability characteristics described above. See, e.g., Table 61.

Preferably, the amphetamine prodrug remains inactive until oraladministration releases the amphetamine. Without being bound by theory,it is believed that the amphetamine prodrug is inactive because theattachment of the chemical moiety reduces binding between theamphetamine and its biological target sites (e.g., human dopamine(“DAT”) and norepinephrine (“NET”) transporter sites). (See Hoebel, B.G., L. Hernandez, et al., “Microdialysis studies of brainnorepinephrine, serotonin, and dopamine release during ingestivebehavior, Theoretical and clinical implications.” Ann NY Acad Sci 575:171-91 (1989)). The chemical moiety attachment may reduce bindingbetween amphetamine and DAT and/or NET in part because the amphetamineprodrug cannot cross the blood-brain barrier. The amphetamine prodrug isactivated by oral administration, that is, the amphetamine is releasedfrom the chemical moiety by hydrolysis, e.g., by enzymes in the stomach,intestinal tract, or blood serum. Because oral administrationfacilitates activation, activation is reduced when the amphetamineprodrug is administered via parenteral routes often employed by illegalusers.

Further, it is believed that the amphetamine prodrug is resistant toabuse and/or overdose due to a natural gating mechanism at the site ofhydrolysis, namely the gastrointestinal tract. This gating mechanism isthought to allow the release of therapeutic amounts of amphetamine fromthe amphetamine prodrug, but limit the release of higher amounts ofamphetamine.

In another embodiment, the toxicity of the amphetamine prodrug issubstantially lower than that of the unbound amphetamine. For example,in a preferred embodiment, the acute toxicity is 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold less, orincrements therein less lethal than oral administration of unboundamphetamine.

Preferably, the amphetamine prodrug provides a serum release curve thatdoes not increase above amphetamine's toxicity level when administeredat higher than therapeutic doses. The amphetamine prodrug may exhibit areduced rate of amphetamine absorption and/or an increased rate ofclearance compared to the free amphetamine. The amphetamine prodrug mayalso exhibit a steady-state serum release curve. Preferably, theamphetamine prodrug provides bioavailability but prevents C_(max)spiking or increased blood serum concentrations. Pharmacokineticparameters are described in the Examples below, particularly theclinical pharmacokinetic Examples. In one embodiment, the amphetamineprodrug provides similar pharmacological activity to the clinicallymeasured pharmacokinetic activity of L-lysine-d-amphetamine dimesylate.For example, the pharmacological parameters (AUC, C_(max), T_(max),C_(min), and/or t_(1/2)) are preferably within 80% to 125%, 80% to 120%,85% to 125%, 90% to 110%, or increments therein, of the given values. Itshould be recognized that the ranges can, but need not be symmetrical,e.g., 85% to 105%. For the pediatric study, the pharmacokineticparameters of d-amphetamine released from L-lysine-d-amphetaminedimesylate are listed in Table 72.

The amphetamine prodrug may exhibit delayed and/or sustained releasecharacteristics. Delayed release prevents rapid onset of pharmacologicaleffects, and sustained release is a desirable feature for particulardosing regimens, e.g., once a day regimens. The amphetamine prodrug mayachieve the release profile independently. Alternatively, theamphetamine prodrug may be pharmaceutically formulated to enhance orachieve such a release profile. It may be desirable to reduce the amountof time until onset of pharmacological effect, e.g., by formulation withan immediate release product.

Accordingly, the invention also provides methods comprising providing,administering, prescribing, or consuming an amphetamine prodrug. Theinvention also provides pharmaceutical compositions comprising anamphetamine prodrug. The formulation of such a pharmaceuticalcomposition can optionally enhance or achieve the desired releaseprofile.

In one embodiment, the invention provides methods for treating a patientcomprising administering a therapeutically effective amount of anamphetamine prodrug, i.e., an amount sufficient to prevent, ameliorate,and/or eliminate the symptoms of a disease. These methods can be used totreat any disease that may benefit from amphetamine-type drugsincluding, but not limited to: attention deficit disorders, e.g., ADDand ADHD, and other learning disabilities; obesity; Alzheimer's disease,amnesia, and other memory disorders and impairments; fibromyalgia;fatigue and chronic fatigue; depression; epilepsy; obsessive compulsivedisorder (OCD); oppositional defiant disorder (ODD); anxiety; resistantdepression; stroke rehabilitation; Parkinson's disease; mood disorder;schizophrenia; Huntington's disorder; dementia, e.g., AIDS dementia andfrontal lobe dementia; movement disfunction; apathy; Pick's disease;Creutzfeldt-Jakob disease, sleep disorders, e.g., narcolepsy, cataplexy,sleep paralysis, and hypnagogic hallucinations; conditions related tobrain injury or neuronal degeneration, e.g., multiple sclerosis,Tourette's syndrome, and impotence; and nicotine dependence andwithdrawal. Preferred indications include ADD, ADHD, narcolepsy, andobesity, with ADHD being most preferred.

The methods of treatment include combination therapies which furthercomprise administering one or more therapeutic agents in addition toadministering an amphetamine prodrug. The active ingredients can beformulated into a single dosage form, or they can be formulated togetheror separately among multiple dosage forms. The active ingredients can beadministered simultaneously or sequentially in any order. Exemplarycombination therapies include the administration of the drugs listed inTable 1:

TABLE 1 Exemplary drug therapies contemplated for use in combinationwith an amphetamine prodrug Condition Exemplary drug class Specificexemplary drugs ADHD Amphetamine Ritalin ®, Dexedrine ®, Adderall ®,Cylert ®, Clonidine, Guanfacine Alzheimer's Reminyl ®, Cognex ®, diseaseAricept ®, Exelon ®, Akatinol ®, Neotropin, Eldepryl ®, Estrogen,Clioquinol, Ibuprofen, Ginko Biloba Anxiety Antidepressant Elavil,Asendin ®, (SSRI, Wellbutrin ®, Tegretol ®, benzodiazepine, Anafranil ®,Norpramine ®, MAOI), anxiolytic Adapin ®, Sinequan ®, Tofranil ®,Epitol ®, Janimire ®, Pamelor ®, Ventyl ®, Aventyl ®, Surmontil ®,Prozac ®, Luvox ®, Serzone ®, Paxil ®, Zoloft ®, Effexor ®, Xanax ®,Librium ®, Klonopin ®, Valium ®, Zetran ®, Valrelease ®, Dalmane ®,Ativan ®, Alzapam ®, Serax ®, Halcion ®, Aurorix ®, Manerix ®, Nardil ®,Parnate ®. Apathy Amisulpride, Olanzapine, Visperidone, Quetiapine,Clozapine, Zotepine Cataplexy Xyrem ® Dementia Thioridazine,Haloperidol, Risperidone, Cognex ®, Aricept ®, Exelon ® DepressionAntidepressant Fluoxetine (e.g., Prozac ®), Zoloft ®, Paxil ®,Reboxetine, Wellbutrin ®, Olanzapine, Elavil ®, Totranil ®, Pamelor ®,Nardil ®, Parnate ®, Desyrel ®, Effexor ® Fatigue BenzodiazepineAnaprox ®, Naprosen, Prozac ®, Zoloft ®, Paxil ®, Effexor ®, Desyrel ®Fibromyalgia Non-steroidal Dilantin ®, Carbatrol ®, anti-inflammatoryEpitol ®, Tegretol ®, drugs Depacon ®, Depakote ®, Norpramin ®,Aventyl ®, Pamelor ®, Elavil ®, Enovil ®, Adapin ®, Sinequan ®,Zonalon ® Hallucinations Clozapine, Risperidone, Zyprexa ®, Seroquel ®Huntington's Haloperidol, Clonzepam disorder Narcolepsy Modafinil (e.g.,Provigil ®), Dexedrine ®, Ritalin ® Mood disorder Thorazine ®, Haldol ®,Navane ®, Mellaril ®, Clozaril ®, Risperidone (e.g., Risperdal ®),Olanzapine (e.g., Zyprexa ®), Clozapine Obsessive- SSRI Anafranil ®,Prozac ®, compulsive Zoloft ®, Paxil ®, Luvox ® disorder (OCD)Oppositional Clonidine, Risperidone, defiant Zyprexa ®, Wellbutrin ®,disorder (ODD) Parkinson's Levodopa, Parlodel ®, disease Permax ®,Mirapex ® Schizophrenia Clozapine, Zyprexa ®, Seroquel ®, andRisperdal ® Sleep paralysis Perocet ®, Vicodin ®, Lorcet ®

A “composition” refers broadly to any composition containing one or moreamphetamine prodrugs. The composition can comprise a dry formulation, anaqueous solution, or a sterile composition. Compositions comprising thecompounds described herein may be stored in freeze-dried form and may beassociated with a stabilizing agent such as a carbohydrate. In use, thecomposition may be deployed in an aqueous solution containing salts,e.g., NaCl, detergents such as sodium dodecyl sulfate (SDS), and othercomponents.

In one embodiment, the amphetamine prodrug itself exhibits a sustainedrelease profile. Thus, the invention provides a pharmaceuticalcomposition exhibiting a sustained release profile due to theamphetamine prodrug.

In another embodiment, a sustained release profile is enhanced orachieved by including a hydrophilic polymer in the pharmaceuticalcomposition. Suitable hydrophilic polymers include, but are not limitedto, natural or partially or totally synthetic hydrophilic gums such asacacia, gum tragacanth, locust bean gum, guar gum, and karaya gum;cellulose derivatives such as methyl cellulose, hydroxymethyl cellulose,hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, and carboxymethyl cellulose; proteinaceous substances such asagar, pectin, carrageen, and alginates; hydrophilic polymers such ascarboxypolymethylene; gelatin; casein; zein; bentonite; magnesiumaluminum silicate; polysaccharides; modified starch derivatives; andother hydrophilic polymers known in the art. Preferably, the hydrophilicpolymer forms a gel that dissolves slowly in aqueous acidic mediathereby allowing the amphetamine prodrug to diffuse from the gel in thestomach. Then when the gel reaches the higher pH medium of theintestines, the hydrophilic polymer dissolves in controlled quantitiesto allow further sustained release. Preferred hydrophilic polymers arehydroxypropyl methylcelluloses such as Methocel ethers, e.g., MethocelE10M® (Dow Chemical Company, Midland, Mich.). One of ordinary skill inthe art would recognize a variety of structures, such as beadconstructions and coatings, useful for achieving particular releaseprofiles. See, e.g., U.S. Pat. No. 6,913,768.

In addition to the amphetamine prodrug, the pharmaceutical compositionsof the invention further comprise one or more pharmaceutical additives.Pharmaceutical additives include a wide range of materials including,but not limited to diluents and bulking substances, binders andadhesives, lubricants, glidants, plasticizers, disintegrants, carriersolvents, buffers, colorants, flavorings, sweeteners, preservatives andstabilizers, and other pharmaceutical additives known in the art. Forexample, in a preferred embodiment, the pharmaceutical compositioncomprises magnesium stearate. In another preferred embodiment, thepharmaceutical composition comprises microcrystalline cellulose (e.g.,Avicel® PH-102), croscarmellose sodium, and magnesium stearate. See,e.g., Table 62.

Diluents increase the bulk of a dosage form and may make the dosage formeasier to handle. Exemplary diluents include, but are not limited to,lactose, dextrose, saccharose, cellulose, starch, and calcium phosphatefor solid dosage forms, e.g., tablets and capsules; olive oil and ethyloleate for soft capsules; water and vegetable oil for liquid dosageforms, e.g., suspensions and emulsions. Additional suitable diluentsinclude, but are not limited to, sucrose, dextrates, dextrin,maltodextrin, microcrystalline cellulose (e.g., Avicel®), microfinecellulose, powdered cellulose, pregelatinized starch (e.g., Starch1500®), calcium phosphate dihydrate, soy polysaccharide (e.g.,Emcosoy®), gelatin, silicon dioxide, calcium sulfate, calcium carbonate,magnesium carbonate, magnesium oxide, sorbitol, mannitol, kaolin,polymethacrylates (e.g., Eudragit®), potassium chloride, sodiumchloride, and talc. A preferred diluent is microcrystalline cellulose(e.g., Avicel® PH-102). Preferred ranges for the amount of diluent byweight percent include about 40% to about 90%, about 50% to about 85%,about 55% to about 80%, about 50% to about 60%, and increments therein.

In embodiments where the pharmaceutical composition is compacted into asolid dosage form, e.g., a tablet, a binder can help the ingredientshold together. Binders include, but are not limited to, sugars such assucrose, lactose, and glucose; corn syrup; soy polysaccharide, gelatin;povidone (e.g., Kollidon®, Plasdone®); Pullulan; cellulose derivativessuch as microcrystalline cellulose, hydroxypropylmethyl cellulose (e.g.,Methocel®), hydroxypropyl cellulose (e.g., Klucel®), ethylcellulose,hydroxyethyl cellulose, carboxymethylcellulose sodium, andmethylcellulose; acrylic and methacrylic acid co-polymers; carbomer(e.g., Carbopol®); polyvinylpolypyrrolidine, polyethylene glycol(Carbowax®); pharmaceutical glaze; alginates such as alginic acid andsodium alginate; gums such as acacia, guar gum, and arabic gums;tragacanth; dextrin and maltodextrin; milk derivatives such as whey;starches such as pregelatinized starch and starch paste; hydrogenatedvegetable oil; and magnesium aluminum silicate.

For tablet dosage forms, the pharmaceutical composition is subjected topressure from a punch and dye. Among other purposes, a lubricant canhelp prevent the composition from sticking to the punch and dyesurfaces. A lubricant can also be used in the coating of a coated dosageform. Lubricants include, but are not limited to, magnesium stearate,calcium stearate, zinc stearate, powdered stearic acid, glycerylmonostearate, glyceryl palmitostearate, glyceryl behenate, silica,magnesium silicate, colloidal silicon dioxide, titanium dioxide, sodiumbenzoate, sodium lauryl sulfate, sodium stearyl fumarate, hydrogenatedvegetable oil, talc, polyethylene glycol, and mineral oil. A preferredlubricant is magnesium stearate. The amount of lubricant by weightpercent is preferably less than about 5%, more preferably 4%, 3%, 2%,1.5%, 1%, or 0.5%, or increments therein.

Glidants can improve the flowability of non-compacted solid dosage formsand can improve the accuracy of dosing. Glidants include, but are notlimited to, colloidal silicon dioxide, fumed silicon dioxide, silicagel, talc, magnesium trisilicate, magnesium or calcium stearate,powdered cellulose, starch, and tribasic calcium phosphate.

Plasticizers include both hydrophobic and hydrophilic plasticizers suchas, but not limited to, diethyl phthalate, butyl phthalate, diethylsebacate, dibutyl sebacate, triethyl citrate, acetyltriethyl citrate,acetyltributyl citrate, cronotic acid, propylene glycol, castor oil,triacetin, polyethylene glycol, propylene glycol, glycerin, andsorbitol. Plasticizers are particularly useful for pharmaceuticalcompositions containing a polymer and in soft capsules and film-coatedtablets. In one embodiment, the plasticizer facilitates the release ofthe amphetamine prodrug from the dosage form.

Disintegrants can increase the dissolution rate of a pharmaceuticalcomposition. Disintegrants include, but are not limited to, alginatessuch as alginic acid and sodium alginate, carboxymethylcellulosecalcium, carboxymethylcellulose sodium (e.g., Ac-Di-Sol®, Primellose®),colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g.,Kollidon®, Polyplasdone®), polyvinylpolypyrrolidine (Plasone-XL®), guargum, magnesium aluminum silicate, methyl cellulose, microcrystallinecellulose, polacrilin potassium, powdered cellulose, starch,pregelatinized starch, sodium starch glycolate (e.g., Explotab®,Primogel®). Preferred disintegrants include croscarmellose sodium andmicrocrystalline cellulose (e.g., Avicel® PH-102). Preferred ranges forthe amount of disintegrant by weight percent include about 1% to about10%, about 1% to about 5%, about 2% to about 3%, and increments therein.

In embodiments where the pharmaceutical composition is formulated for aliquid dosage form, the pharmaceutical composition may include one ormore solvents. Suitable solvents include, but are not limited to, water;alcohols such as ethanol and isopropyl alcohol; methylene chloride;vegetable oil; polyethylene glycol; propylene glycol; and glycerin.

The pharmaceutical composition can comprise a buffer. Buffers include,but are not limited to, lactic acid, citric acid, acetic acid, sodiumlactate, sodium citrate, and sodium acetate.

Any pharmaceutically acceptable colorant can be used to improveappearance or to help identify the pharmaceutical composition. See 21C.F.R., Part 74. Exemplary colorants include D&C Red No. 28, D&C YellowNo. 10, FD&C Blue No. 1, FD&C Red No. 40, FD&C Green #3, FD&C Yellow No.6, and edible inks. Preferred colors for gelatin capsules include white,medium orange, and light blue.

Flavorings improve palatability and may be particularly useful forchewable tablet or liquid dosage forms. Flavorings include, but are notlimited to maltol, vanillin, ethyl vanillin, menthol, citric acid,fumaric acid, ethyl maltol, and tartaric acid. Sweeteners include, butare not limited to, sorbitol, saccharin, sodium saccharin, sucrose,aspartame, fructose, mannitol, and invert sugar.

The pharmaceutical compositions of the invention can also include one ormore preservatives and/or stabilizers to improve storagability. Theseinclude, but are not limited to, alcohol, sodium benzoate, butylatedhydroxy toluene, butylated hydroxyanisole, and ethylenediaminetetraacetic acid.

Other pharmaceutical additives include gelling agents such as colloidalclays; thickening agents such as gum tragacanth and sodium alginate;wetting agents such as lecithin, polysorbates, and laurylsulphates;humectants; antioxidants such as vitamin E, caronene, and BHT;adsorbents; effervescing agents; emulsifying agents, viscosity enhancingagents; surface active agents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate, triethanolamine, polyoxyethylene sorbitan,poloxalkol, and quaternary ammonium salts; and other miscellaneousexcipients such as lactose, mannitol, glucose, fructose, xylose,galactose, sucrose, maltose, xylitol, sorbitol, chloride, sulfate andphosphate salts of potassium, sodium, and magnesium.

The pharmaceutical compositions can be manufactured according to anymethod known to those of skill in the art of pharmaceutical manufacturesuch as, for example, wet granulation, dry granulation, encapsulation,direct compression, slugging, etc. For instance, a pharmaceuticalcomposition can be prepared by mixing the amphetamine prodrug with oneor more pharmaceutical additives with an aliquot of liquid, preferablywater, to form a wet granulation. The wet granulation can be dried toobtain granules. The resulting granulation can be milled, screened, andblended with various pharmaceutical additives such as water-insolublepolymers and additional hydrophilic polymers. In one embodiment, anamphetamine prodrug is mixed with a hydrophilic polymer and an aliquotof water, then dried to obtain granules of amphetamine prodrugencapsulated by hydrophilic polymer.

After granulation, the pharmaceutical composition is preferablyencapsulated, e.g., in a gelatin capsule. The gelatin capsule cancontain, for example, kosher gelatin, titanium dioxide, and optionalcolorants. Alternatively, the pharmaceutical composition can betableted, e.g., compressed and optionally coated with a protectivecoating that dissolves or disperses in gastric juices.

The pharmaceutical compositions of the invention can be administered bya variety of dosage forms. Any biologically-acceptable dosage form knownin the art, and combinations thereof, are contemplated. Examples ofpreferred dosage forms include, without limitation, tablets includingchewable tablets, film-coated tablets, quick dissolve tablets,effervescent tablets, multi-layer tablets, and bi-layer tablets;caplets; powders including reconstitutable powders; granules;dispersible granules; particles; microparticles; capsules including softand hard gelatin capsules; lozenges; chewable lozenges; cachets; beads;liquids; solutions; suspensions; emulsions; elixirs; and syrups.

The pharmaceutical composition is preferably administered orally. Oraladministration permits the maximum release of amphetamine, providessustained release of amphetamine, and maintains abuse resistance.Preferably, the amphetamine prodrug releases the amphetamine over a moreextended period of time as compared to administering unboundamphetamine.

Oral dosage forms can be presented as discrete units, such as capsules,caplets, or tablets. In a preferred embodiment, the invention provides asolid oral dosage form comprising an amphetamine prodrug that is smallerin size compared to a solid oral dosage form containing atherapeutically equivalent amount of unbound amphetamine. In oneembodiment, the oral dosage form comprises a gelatin capsule of size 2,size 3, or smaller (e.g., size 4). The smaller size of the amphetamineprodrug dosage forms promotes ease of swallowing.

Soft gel or soft gelatin capsules may be prepared, for example, bydispersing the formulation in an appropriate vehicle (e.g., vegetableoil) to form a high viscosity mixture. This mixture then is encapsulatedwith a gelatin based film. The industrial units so formed are then driedto a constant weight.

Chewable tablets can be prepared by mixing the amphetamine prodrug withexcipients designed to form a relatively soft, flavored tablet dosageform that is intended to be chewed. Conventional tablet machinery andprocedures (e.g., direct compression, granulation, and slugging) can beutilized.

Film-coated tablets can be prepared by coating tablets using techniquessuch as rotating pan coating methods and air suspension methods todeposit a contiguous film layer on a tablet.

Compressed tablets can be prepared by mixing the amphetamine prodrugwith excipients that add binding qualities. The mixture can be directlycompressed, or it can be granulated and then compressed.

The pharmaceutical compositions of the invention can alternatively beformulated into a liquid dosage form, such as a solution or suspensionin an aqueous or non-aqueous liquid. The liquid dosage form can be anemulsion, such as an oil-in-water liquid emulsion or a water-in-oilliquid emulsion. The oils can be administered by adding the purified andsterilized liquids to a prepared enteral formula, which then is placedin the feeding tube of a patient who is unable to swallow.

For oral administration, fine powders or granules containing diluting,dispersing, and/or surface-active agents can be presented in a draught,in water or a syrup, in capsules or sachets in the dry state, in anon-aqueous suspension wherein suspending agents may be included, or ina suspension in water or a syrup. Liquid dispersions for oraladministration can be syrups, emulsions, or suspensions. The syrups,emulsions, or suspensions can contain a carrier, for example, a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, saccharose, saccharose with glycerol, mannitol,sorbitol, and polyvinyl alcohol.

The dose range of the amphetamine prodrug for humans will depend on anumber of factors including the age, weight, and condition of thepatient. Tablets and other dosage forms provided in discrete units cancontain a daily dose, or an appropriate fraction thereof, of one or moreamphetamine prodrugs. The dosage form can contain a dose of about 2.5 mgto about 500 mg, about 10 mg to about 250 mg, about 10 mg to about 100mg, about 25 mg to about 75 mg, or increments therein of one or more ofthe amphetamine prodrugs. In a preferred embodiment, the dosage formcontains 30 mg, 50 mg, or 70 mg of an amphetamine prodrug.

The dosage form can utilize any one or any combination of known releaseprofiles including, but not limited to immediate release, extendedrelease, pulse release, variable release, controlled release, timedrelease, sustained release, delayed release, and long acting.

The pharmaceutical compositions of the invention can be administered ina partial, i.e., fractional dose, one or more times during a 24 hourperiod. Fractional, single, double, or other multiple doses can be takensimultaneously or at different times during a 24 hour period. The dosescan be uneven doses with regard to one another or with regard to theindividual components at different administration times. Preferably, asingle dose is administered once daily. The dose can be administered ina fed or fasted state.

The dosage units of the pharmaceutical composition can be packagedaccording to market need, for example, as unit doses, rolls, bulkbottles, blister packs, and so forth. The pharmaceutical package, e.g.,blister pack, can further include or be accompanied by indicia allowingindividuals to identify the identity of the pharmaceutical composition,the prescribed indication (e.g., ADHD), and/or the time periods (e.g.,time of day, day of the week, etc.) for administration. The blister packor other pharmaceutical package can also include a second pharmaceuticalproduct for combination therapy.

It will be appreciated that the pharmacological activity of thecompositions of the invention can be demonstrated using standardpharmacological models that are known in the art. Furthermore, it willbe appreciated that the inventive compositions can be incorporated orencapsulated in a suitable polymer matrix or membrane for site-specificdelivery, or can be functionalized with specific targeting agentscapable of effecting site specific delivery. These techniques, as wellas other drug delivery techniques, are well known in the art.

Any feature of the above-describe embodiments can be used in combinationwith any other feature of the above-described embodiments.

In order to facilitate a more complete understanding of the invention,Examples are provided below. However, the scope of the invention is notlimited to specific embodiments disclosed in these Examples, which arefor purposes of illustration only.

EXAMPLES

The following abbreviations are used in the Examples and throughout thepatent:

-   -   Lys-Amp=L-lysine-d-amphetamine, Lysine-Amphetamine, K-Amp,        K-amphetamine, or 2,6-diaminohexanoic        acid-(1-methyl-2-phenylethyl)-amide, or Lisdexamfetamine    -   Phe-Amp=Phenylalanine-Amphetamine, F-Amp, or        2-amino-3-phenylpropanoic acid-(1-methyl-2-phenylethyl)-amide    -   Ser-Amp=Serine-Amphetamine, S-Amp, or        2-amino-3-hydroxylpropanoic acid-(1-methyl-2-phenylethyl)-amide,    -   Gly₃-Amp=GGG-Amphetamine, GGG-Amp, or        2-amino-N-({[(1-methyl-2-phenyl-ethylcarbornyl)-methyl]-carbornyl}-methyl)-acetamide    -   BOC=t-butyloxycarbonyl    -   CMC=carboxymethylcellulose    -   DIPEA=di-isopropyl ethyl amine    -   mp=melting point    -   NMR=nuclear magnetic resonance    -   OSu=hydroxysuccinimido ester

Throughout the Examples, unless otherwise specified, doses are describedas the amount of d-amphetamine base. Exemplary conversions are providedin Table 2.

TABLE 2 Conversion of d-amphetamine doses (mg) L-lysine-d-amphetaminedimesylate d-amphetamine d-amphetamine sulfate (29.5% d-amphetamine)base (72.8% d-amphetamine) 5.08 1.5 2.06 10.17 3 4.12 20.34 6 8.24 40.6812 16.48 101.69 30 41.21 203.39 60 82.42 25.00 7.375 10.13 75.00 22.12530.39 70.00 20.65 28.37 50.00 14.75 20.26 30.00 8.85 12.16

Example 1 General Synthesis of Peptide Amphetamine Conjugates

Peptide conjugates were synthesized by the general method described inFIG. 1. An iterative approach can be used to identify favorableconjugates by synthesizing and testing single amino acid conjugates, andthen extending the peptide one amino acid at a time to yield dipeptideand tripeptide conjugates, etc. The parent single amino acid prodrugcandidate may exhibit more or less desirable characteristics than itsdi- or tripeptide offspring candidates. The iterative approach canquickly suggest whether peptide length influences bioavailability.

General Synthesis of Single Amino Acid Amphetamine Conjugates

To a solution of a protected amino acid succinimidyl ester (2.0 eq) in1,4-dioxane (30 mL) was added d-amphetamine sulfate (1.0 eq) and NMM(4.0 eq). The resulting mixture was allowed to stir for 20 h at 20° C.Water (10 mL) was added, and the solution was stirred for 10 minutesprior to removing solvents under reduced pressure. The crude product wasdissolved in EtOAc (100 mL) and washed with 2% AcOH_(aq) (3×100 mL),saturated NaHCO₃ solution (2×50 mL), and brine (1×100 mL). The organicextract was dried over MgSO₄, filtered, and evaporated to dryness toafford the protected amino acid amphetamine conjugate. This intermediatewas directly deprotected by adding 4 N HCl in 1,4-dioxane (20 mL). Thesolution was stirred for 20 h at 25° C. The solvent was evaporated, andthe product dried in vacuum to afford the corresponding amino acidamphetamine hydrochloride conjugate. The syntheses of exemplary singleamino acid conjugates are depicted in FIG. 2-FIG. 6.

General Synthesis of Dipeptide Amphetamine Conjugates

To a solution of a protected dipeptide succinimidyl ester (1.0 eq) in1,4-dioxane was added amphetamine sulfate (2.0 eq) and NMM (4.0 eq). Theresulting mixture was stirred for 20 h at 25° C. Solvents were removedunder reduced pressure. Saturated NaHCO₃ solution (20 mL) was added, andthe suspension was stirred for 30 min. IPAC (100 mL) was added, and theorganic layer was washed with 2% AcOH_(aq) (3×100 mL) and brine (2×100mL). The organic extract was dried over Na₂SO₄, and the solvent wasevaporated to dryness to yield the protected dipeptide amphetamineconjugate. The protected dipeptide conjugate was directly deprotected byadding 4 N HCl in 1,4-dioxane (20 mL), and the solution stirred for 20 hat 25° C. The solvent was evaporated, and the product was dried invacuum to afford the corresponding dipeptide amphetamine hydrochlorideconjugate.

General Synthesis of Tripeptide Amphetamine Conjugates

An amino acid conjugate was synthesized and deprotected according to thegeneral procedure described above. To a solution of the amino acidamphetamine hydrochloride (1.0 eq) in dioxane (20 mL) was added NMM (5.0eq) and a protected dipeptide succinate (1.05 eq). The solution wasstirred for 20 h at 25° C. The solvent was removed under reducedpressure. Saturated NaHCO₃ solution (20 mL) was added, and thesuspension was stirred for 30 min. IPAC (100 mL) was added, and theorganic layer was washed with 2% AcOH_(aq) (3×100 mL) and brine (2×100mL). The organic extract was dried over Na₂SO₄, and the solvent wasevaporated to dryness to yield the protected tripeptide amphetamine.Deprotection was directly carried out by adding 4 N HCl in 1,4-dioxane(20 mL). The mixture was stirred for 20 h at 25° C., the solvent wasevaporated, and the product was dried in vacuum to afford the respectivetripeptide amphetamine hydrochloride conjugate.

The hydrochloride conjugates required no further purification, but manyof the deprotected hydrochloride salts were hygroscopic and requiredspecial handling during analysis and subsequent in vivo testing.

Example 2 Synthesis of L-lysine-d-amphetamine

L-lysine-d-amphetamine was synthesized by the following methods.

a. Preparation of HCl salt (see FIG. 3)

i. Coupling

Reagents MW Weight mmoles Molar Equivalents d-amphetamine free 135.24.75 g 35.13 1 base Boc-Lys(Boc)-OSu 443.5 15.58 g 35.13 1Di-iPr-Et-Amine 129 906 mg 7.03 0.2, d = 0.74, 1.22 mL 1,4-Dioxane — 100mL — —

To a solution of Boc-Lys(Boc)-OSu (15.58 g, 35.13 mmol) in dioxane (100mL) under an inert atmosphere was added d-amphetamine free base (4.75 g,35.13 mmol) and DIPEA (0.9 g, 1.22 mL, 7.03 mmol). The resulting mixturewas allowed to stir at room temperature overnight. Solvent and excessbase were then removed using reduced pressure evaporation. The crudeproduct was dissolved in ethyl acetate and loaded on to a flash column(7 cm wide, filled to 24 cm with silica) and eluted with ethyl acetate.The product was isolated, the solvent reduced by rotary evaporation, andthe purified protected amide was dried by high-vac to obtain a whitesolid. ¹H NMR (DMSO-d₆) δ 1.02-1.11 (m, 2H, Lys γ-CH₂), δ 1.04 (d, 3H,Amp α-CH₃), δ 1.22-1.43 (m, 4H, Lys-β and δ-CH₂), δ 1.37 (18H, Boc,6×CH₃), δ 2.60-2.72 (2H, Amp CH₂), δ 3.75-3.83, (m, 1H, Lys α-H) δ3.9-4.1 (m, 1H, Amp α-H), δ 6.54-6.61 (d, 1H, amide NH), δ 6.7-6.77 (m,1H, amide NH), δ 7.12-7.29 (m, 5H, ArH), δ 7.65-7.71 (m, 1, amide NH);mp=86-88° C.

ii. Deprotection

Molar Reagents MW Weight mmoles Equivalents 4M HCl in dioxane 4 mmol/mL50 mL 200 6.25 Boc-Lys(Boc)-Amp 463.6 14.84 g 32 1 1,4-Dioxane — 50 mL ——

The protected amide was dissolved in 50 mL of anhydrous dioxane andstirred while 50 mL (200 mmol) of 4M HCl/dioxane was added and stirredat room temperature overnight. The solvents were then reduced by rotaryevaporation to afford a viscous oil. Addition of 100 mL MeOH followed byrotary evaporation resulted in a golden colored solid material that wasfurther dried by storage at room temperature under high vacuum. ¹H NMR(DMSO-d₆) δ 0.86-1.16 (m, 2H, Lys γ-CH₂), δ 1.1 (d, 3H, Amp α-CH₃), δ1.40-1.56 (m, 4H, Lys-β and δ-CH₂), δ 2.54-2.78 (m, 2H, Amp CH₂, 2H, Lysε-CH₂), 3.63-3.74 (m, 1H, Lys α-H), δ 4.00-4.08 (m, 1H, Amp α-H), δ7.12-7.31 (m, 5H, Amp ArH), δ 8.13-8.33 (d, 3H, Lys amine) δ 8.70-8.78(d, 1H, amide NH); mp=120-122° C.

b. Preparation of Mesylate Salt (and See FIG. 2)

Similarly, the mesylate salt of the peptide conjugate can be prepared byusing methanesulfonic acid in the deprotection step as described infurther detail below.

i. Coupling

A 72-L round-bottom reactor was equipped with a mechanical stirrer,digital thermocouple, and addition funnel and purged with nitrogen. Thevessel was charged with Boc-Lys(Boc)-OSu (3.8 kg, 8.568 mol, 1.0 eq) and1,4-dioxane (20.4 L), and the resulting turbid solution was stirred at20±5° C. for 10 min. To the mixture was added N-methylmorpholine (950 g,9.39 mol, 1.09 eq) over a period of 1 min, and the mixture was stirredfor 10 min. To the slightly turbid reaction mixture was then added asolution of dextro-amphetamine (1.753 kg, 12.96 mol, 1.51 eq) in1,4-dioxane (2.9 L) over a period of 30 min, while cooling the reactorexternally with an ice/water bath. The internal temperature was keptbelow 25° C. during the addition. At the end of the addition, a thickwhite precipitate appeared. The addition funnel was rinsed with1,4-dioxane (2.9 L) into the reactor, and the reaction mixture wasstirred at 22±3° C. TLC monitoring 30 min. after completed additionshowed no more remaining Boc-Lys(Boc)-Osu, and the reaction was quenchedwith DI H₂O (10 L). The mixture was stirred for 1 h at ambienttemperature and then concentrated under reduced pressure to afford adense, white solid.

For the extractions, two solutions were prepared: an acetic acid/saltsolution: NaCl (15 kg) and glacial acetic acid (2 kg) in DI H₂O (61 L),and a bicarbonate solution: NaHCO₃ (1.5 kg) in DI H₂O (30 L).

The solid was re-dissolved in IPAC (38 L) and acetic acid/salt solution(39 kg) and transferred into a 150-L reactor. The layers were mixed for10 min. and then allowed to separate. The organic layer was drained andwashed with another portion (39 kg) of acetic acid/salt solution,followed by a wash with bicarbonate solution (31.5 kg). All phaseseparations occurred within 5 min. To the organic solution was thenadded silica-gel (3.8 kg; Silica-gel 60). The resulting slurry wasstirred for 45 min. and then filtered through filter paper. Thefilter-cake was washed with IPAC (5×7.6 L). The filtrate and washes wereanalyzed by TLC, and it was determined that all contained product. Thefiltrate and washes were combined and concentrated under reducedpressure to afford the crude product as a white solid.

ii. Deprotection

A 45-L carboy was charged with di-Boc-Lys-Amp (3.63 kg, 7.829 mol) and1,4-dioxane (30.8 L, 8.5 vol), and the mixture was stirred rapidly undernitrogen for 30 min. The resulting solution was filtered, and thefilter-cake was rinsed with 1,4-dioxane (2×1.8 L).

The filtrates were then transferred into a 72-L round-bottom flask,which was equipped with a mechanical stirrer, digital thermocouple,nitrogen inlet and outlet, and 5 L addition funnel. The temperature ofthe reaction mixture was regulated at 21±3° C. with a water bath. To theclear, slightly yellow solution was added methanesulfonic acid (3.762kg, 39.15 mol, 5 eq) over a period of 1 h while keeping the internaltemperature at 21±3° C. Approximately 1 h after completed addition, awhite precipitate started to appear. The mixture was stirred at ambienttemperature for 20.5 h, after which HPLC monitoring showed thedisappearance of all starting material. The mixture was filtered throughfilter-paper, and the reaction vessel was rinsed with 1,4-dioxane (3.6L, 1 vol). The filter-cake was washed with dioxane (3×3.6 L) and driedwith a rubber dam for 1 h. The material was then transferred to dryingtrays and dried in a vacuum oven at 55° C. for ˜90 h. This affordedLys-Amp dimesylate [3.275 kg, 91.8% yield; >99% (AUC)] as a white solid.

Example 3 Synthesis of Ser-Amp

Ser-Amp was synthesized by a similar method (see FIG. 4) except theamino acid starting material was Boc-Ser(O-tBu)-OSu and the deprotectionwas done using a solution of trifluoroacetic acid instead of HCl.

Example 4 Synthesis of Phe-Amp

Phe-Amp was synthesized by a similar method (see FIG. 5) except theamino acid starting material was Boc-Phe-OSu.

Phe-Amp hydrochloride: hygroscopic; ¹H NMR (400 MHz, DMSO-d₆): δ 8.82(d, J=8.0 Hz, 1H), 8.34 (bs, 3H), 7.29-7.11 (m, 10H), 3.99 (m, 2H), 2.99(dd, J=13.6, 6.2 Hz, 1H), 2.88 (dd, J=13.6, 7.2 Hz, 1H), 2.64 (dd,J=13.2, 7.6 Hz, 1H), 2.53 (m, 1H), 1.07 (d, J=6.4 Hz, 3H); ¹³C NMR (100MHz, DMSO-d₆): δ 167.31, 139.27, 135.49, 130.05, 129.66, 128.78, 128.61,127.40, 126.60, 53.83, 47.04, 42.15, 37.27, 20.54; HRMS: (ESI) forC₁₈H₂₃N₂O (M+H)⁺: calcd, 283.1810: found, 283.1806.

Example 5 Synthesis of Gly₃-Amp

Gly₃-Amp was synthesized by a similar method (see FIG. 6) except theamino acid starting material was Boc-GGG-OSu.

Gly₃-Amp hydrochloride: mp 212-214° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 7.28(m, 5H), 3.96 (m, 1H), 3.86 (m, 2H), 3.66 (m, 4H), 2.76 (m, 1H), 2.75(m, 1H), 1.02 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.91,168.14, 166.85, 139.45, 129.60, 128.60, 126.48, 46.60, 42.27, 20.30.HRMS: (ESI) for C₁₅H₂₂N₄O₃Na (M+Na)⁺: calcd, 329.1590: found, 329.1590.

Example 6 Pharmacokinetics of L-lysine-d-amphetamine diHCl Compared tod-amphetamine Sulfate (ELISA Analysis)

Male Sprague-Dawley rats were provided water ad libitum, fastedovernight, and dosed by oral gavage L-lysine-d-amphetamine diHCl ord-amphetamine sulfate. In all studies, doses contained equivalentamounts of d-amphetamine base. Plasma d-amphetamine concentrations weremeasured by ELISA (Amphetamine Ultra, 109319, Neogen, Corporation,Lexington, Ky.). The assay is specific for d-amphetamine with onlyminimal reactivity (0.6%) of the major d-amphetamine metabolite(para-hydroxy-d-amphetamine) occurring. L-lysine-d-amphetamine diHCl wasalso determined to be essentially unreactive in the ELISA (<1%).

Mean (n=4) plasma concentration curves of d-amphetamine orL-lysine-d-amphetamine diHCl are shown in FIG. 7. Extended release wasobserved in all four L-lysine-d-amphetamine diHCl dosed animals, andC_(max) was substantially decreased as compared to animals dosed withd-amphetamine sulfate. Plasma d-amphetamine concentrations of individualanimals for d-amphetamine or L-lysine-d-amphetamine diHCl are shown inTable 3. The mean plasma d-amphetamine concentrations are shown in Table4. The time to peak concentration for L-lysine-d-amphetamine diHCl wassimilar to that of d-amphetamine. Pharmacokinetic parameters for oraladministration of d-amphetamine or L-lysine-d-amphetamine diHCl aresummarized in Table 5.

TABLE 3 Plasma concentrations of d-amphetamine from individual animalsorally administered d-amphetamine or L-lysine-d-amphetamine diHCl (3mg/kg d- amphetamine base) Time d-amphetamine (ng/ml)L-lysine-d-amphetamine (ng/ml) (hours) Rat #1 Rat #2 Rat #3 Rat #4 Rat#1 Rat #2 Rat #3 Rat #4 0.5 144 157 101 115 52 62 74 44 1 152 78 115 7848 72 79 57 1.5 85 97 117 95 42 62 76 53 3 34 45 72 38 61 60 71 43 5 2014 12 15 49 33 44 22 8 3 3 2 2 15 14 12 8

TABLE 4 Mean plasma concentrations of d-amphetamine following oraladministration of d-amphetamine or L-lysine-d-amphetamine Plasmad-amphetamine Concentrations (ng/ml) d-amphetamineL-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0.5 129 25 20 58 1322 1 106 35 33 64 14 22 1.5 99 13 14 58 14 25 3 47 17 36 59 11 19 5 15 424 37 12 32 8 2 1 35 12 3 24

TABLE 5 Pharmacokinetic parameters of d-amphetamine following oraladministration of d-amphetamine or L-lysine-d-amphetamine Mean AUC (0-8h) Percent C_(max) Percent Peak Percent Drug ng · h/mL Amphetamine(ng/ml) Amphetamine (ng/ml) Amphetamine Amphetamine 341 ± 35 100 111 ±27 100 129 100 Lys-Amp 333 ± 66 98  61 ± 13 55 64 50

This example illustrates that when lysine is conjugated to the activeagent amphetamine, the peak levels of amphetamine are decreased whilebioavailability is maintained approximately equal to amphetamine. Thebioavailability of amphetamine released from L-lysine-d-amphetamine issimilar to that of amphetamine sulfate at the equivalent dose; thusL-lysine-d-amphetamine maintains its therapeutic value. The gradualrelease of amphetamine from L-lysine-d-amphetamine and decrease in peaklevels reduce the possibility of overdose.

Example 7 Oral Bioavailability of L-lysine-d-amphetamine Dimesylate atVarious Doses

a. Doses Approximating Therapeutic Human Doses (1.5, 3, and 6 mg/kg)

Mean (n=4) plasma concentration curves of d-amphetamine vs.L-lysine-d-amphetamine are shown for rats orally administered 1.5, 3,and 6 mg/kg in FIG. 8, FIG. 9, and FIG. 10, respectively. Extendedrelease was observed at all three therapeutic doses forL-lysine-d-amphetamine dosed animals. The mean plasma concentrations for1.5, 3, and 6 mg/kg are shown in Table 6, Table 7, and Table 8,respectively. Pharmacokinetic parameters for oral administration ofd-amphetamine vs. L-lysine-d-amphetamine at the various doses aresummarized in Table 9.

TABLE 6 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (1.5 mg/kg) PlasmaAmphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamineHours Mean ±SD CV Mean ±SD CV 0 0 0 0 0 0 0 0.25 103 22 21 31 11 37 0.5126 20 16 51 23 45 1 101 27 27 68 23 34 1.5 116 28 24 72 10 14 3 66 1320 91 5 5 5 40 7 18 75 16 22 8 17 2 15 39 13 34

TABLE 7 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (3 mg/kg) Plasma AmphetamineConcentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean±SD CV Mean ±SD CV 0 0 0 0.25 96 41 43 51 49 97 0.5 107 49 46 36 35 96 1121 17 14 81 44 54 1.5 120 33 27 97 32 33 3 91 30 33 88 13 15 5 62 22 3691 21 23 8 19 6 33 46 16 34

TABLE 8 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (6 mg/kg) Plasma AmphetamineConcentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean±SD CV Mean ±SD CV 0 0 0 0.25 204 14 7 74 38 51 0.5 186 9 5 106 39 37 1167 12 7 133 33 24 1.5 161 24 15 152 22 15 3 111 29 26 157 15 10 5 78 911 134 18 13 8 35 5 15 79 12 15

TABLE 9 Pharmacokinetic parameters of d-amphetamine following oraladministration of d-amphetamine or L-lysine-d-amphetamine 1.5 mg/kg 3mg/kg 6 mg/kg Parameter Amp K-Amp Amp K-Amp Amp K-Amp AUC 481 538 587614 807 1005 (ng · h/ml) Percent 100 112 100 105 100 125 C_(max) (ng/ml)133 93 141 104 205 162 Percent 100 70 100 74 100 79 T_(max) (hours)0.938 3.5 1 1.56 0.563 2.625 Percent 100 373 100 156 100 466

b. Increased doses (12, 30, and 60 mg/kg)

Mean (n=4) plasma concentration curves of d-amphetamine vs.L-lysine-d-amphetamine are shown for rats orally administered 12, 30,and 60 mg/kg. At these higher doses, the bioavailability ofL-lysine-d-amphetamine was markedly decreased as compared tod-amphetamine.

TABLE 10 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (12 mg/kg) Plasma AmphetamineConcentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 530 279 53 53 34 64 0.5 62176 12 99 32 33 1 512 91 18 220 77 35 1.5 519 113 22 224 124 55 3 376 14940 300 153 51 5 314 123 39 293 153 52 8 103 64 63 211 45 22

TABLE 11 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (30 mg/kg) Plasma AmphetamineConcentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 2,036 1,262 62 29 16 54 0.52,583 1,465 57 88 29 34 1 3,162 772 24 328 30 9 1.5 3,445 191 6 368 9927 3 2,620 72 3 620 79 13 5 1,535 21 1 730 169 23 8 164 52 32 NA NA NA

TABLE 12 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration (60 mg/kg) Plasma AmphetamineConcentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 3,721 286  8 169  93 55 0.53,566 560 16 259 138 53 1 3,556 442 12 420 173 41 1.5 4,142 381  9 506169 33 3 NA NA NA 686 222 32 5 NA NA NA 612  67 11 8 NA NA NA 870 NA NA

TABLE 13 Pharmacokinetic parameters of d-amphetamine following oraladministration of d-amphetamine or L-lysine-d-amphetamine 12 mg/kg 30mg/kg 60 mg/kg Parameter Amp K-Amp Amp K-Amp Amp K-Amp AUC (ng · h/ml)2,738 1,958  12,623*  2,387*  5,081**  476** Percent 100 72   100   19  100  9 C_(max) (ng/ml) 621 352  3,726   231 4,101 647 Percent 100 57  100    6   100  16 T_(max) (hours) 0.938 3.5 NA NA NA NA Percent 100373 NA NA NA NA *0-5 h **0-1.5 h

Example 8 Oral Bioavailability of L-lysine-d-amphetamine Dimesylate atVarious Doses Approximating a Range of Therapeutic Human Doses Comparedto a Suprapharmacological Dose

Male Sprague-Dawley rats were provided water ad libitum, fastedovernight, and dosed by oral gavage with 1.5, 3, 6, 12, and 60 mg/kg ofamphetamine sulfate or L-lysine-d-amphetamine containing the equivalentamounts of d-amphetamine. Concentrations of d-amphetamine were measuredby ELISA.

It has been demonstrated that when lysine is conjugated to the activeagent d-amphetamine, the levels of d-amphetamine at 30 minutespost-administration are decreased by approximately 50% over a dosagerange of 1.5 to 12 mg/kg. However, when a suprapharmacological dose (60mg/kg) is given, the levels of d-amphetamine from L-lysine-d-amphetamineonly reached 8% of those seen for d-amphetamine sulfate (See Table 14,Table 15, and FIG. 15). The substantial decrease in oral bioavailabilityat a high dose greatly reduces the abuse potential ofL-lysine-d-amphetamine.

TABLE 14 Levels of d-amphetamine vs. dosage at 0.5 h post dosing withd-amphetamine sulfate Dose mg/kg 1.5 3 6 12 60 ng/ml 0.5 h 109 ± 59 196± 72 294 ± 202 344 ± 126 3239 ± 73 Percent 100 100 100 100 100

TABLE 15 Levels of d-amphetamine vs. dosage at 0.5 h post dosing withL-lysine-d-amphetamine Dose mg/kg 1.5 3 6 12 60 ng/ml 0.5 h 45 ± 10 86 ±26 129 ± 46 172 ± 113 266 ± 18 Percent 41 44 44 50 8

Example 9 Decreased Oral Bioavailability of L-lysine-d-amphetamineDimesylate at a High Dose

An additional oral PK study illustrated in FIG. 16 shows thed-amphetamine blood levels of a 60 mg/kg dose over an 8 h time course.In the case of d-amphetamine, blood levels quickly reached a very highlevel, and 8 of 12 animals either died or were sacrificed due to acutesymptoms of toxicity. Blood levels (Table 16 and Table 17) of animalsadministered L-lysine-d-amphetamine, on the other hand, did not peakuntil 5 hours and reached only a fraction of the levels of the animalsreceiving amphetamine. (Note: Valid data past 3 h for d-amphetaminecould not be determined due to death and sacrifice of animals).

TABLE 16 Mean plasma concentrations of d-amphetamine vs. L-lysine-d-amphetamine following oral administration of a high dose (60 mg/kg)Plasma Amphetamine Concentrations (ng/ml) d-amphetamineL-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0 NA NA NA NA NA NA0.25 2174 907 42 35 17 48 0.5 2643 578 22 81 33 41 1 2828 1319 47 212 3014 1.5 2973 863 29 200 79 40 3  2944* 95  3 440 133 30 5  153* NA NA 565100 18 8  1309** NA NA 410 206 50 *n = 2 **n = 1

TABLE 17 Pharmacokinetic parameters of d-amphetamine vs. L-lysine-d-amphetamine Mean AUC Percent C_(max) Percent Peak Percent Drug ng ·h/ml d-Amp (ng/ml) d-Amp (ng/ml) d-Amp d- 13420 100 3623 100 2973 100amphetamine L-lysine-d- 3,143 39 582 16 565 19 amphetamine

Example 10 Oral Bioavailability of d-amphetamine FollowingAdministration of an Extended Release Formulation (Intact or Crushed) orL-lysine-d-amphetamine Dimesylate

Doses of an extended release formulation of d-amphetamine sulfate(Dexedrine Spansule® capsules, GlaxoSmithKline) were orally administeredto rats as intact capsules or as crushed capsules and compared to a doseof L-lysine-d-amphetamine containing an equivalent amount ofd-amphetamine base (FIG. 20). The crushed capsules showed an increase inC_(max) and AUC_(inf) of 84 and 13 percent, respectively, as compared tointact capsules (Table 18 and Table 19). In contrast, C_(max) andAUC_(inf) of d-amphetamine following administration ofL-lysine-d-amphetamine were similar to that of the intact capsuleillustrating that extended release is inherent to the compound itselfand can not be circumvented by simple manipulation.

TABLE 18 Time-course concentrations of d-amphetamine following oraladministration of extended release Dexedrine Spansule ® capsules,crushed extended release Dexedrine Spansule ® capsules, orL-lysine-d-amphetamine (3 mg/kg d-amphetamine base) Plasma Concentration(ng/ml) Intact Spansule ® Crushed Spansule ® L-lysine-d- Hours CapsuleCapsule amphetamine 0 0 0 0 0.25 32 46 3 0.5 33 85 5 1 80 147 34 1.5 61101 60 3 64 66 76 5 46 39 66 8 34 12 38

TABLE 19 Pharmacokinetic parameters of d-amphetamine following oraladministration of extended release Dexedrine Spansule ® capsules,crushed extended release Dexedrine Spansule ® capsules, orL-lysine-d-amphetamine (3 mg/kg d-amphetamine base) Intact Spansule ®Crushed Spansule ® L-lysine-d- Parameter Capsule Capsule amphetamineAUC_(0-8h) 399 449 434 (ng · h/ml) Percent 100 113 109 C_(max) (ng/ml)80 147 76 Percent 100 184 95 T_(max) (hours) 1 1 3 Percent 100 100 300

This example illustrates the advantage of the invention overconventional controlled release formulations of d-amphetamine.

Example 11 Decreased Intranasal Bioavailability ofL-lysine-d-amphetamine vs. Amphetamine

a. Intranasal (IN) Bioavailability of L-lysine-d-amphetamineHydrochloride

Male Sprague-Dawley rats were dosed by intranasal administration with 3mg/kg of amphetamine sulfate or L-lysine-d-amphetamine hydrochloridecontaining the equivalent amounts of d-amphetamine.L-lysine-d-amphetamine did not release any significant amount ofd-amphetamine into circulation by IN administration. Mean (n=4) plasmaamphetamine concentration curves of amphetamine vs.L-lysine-d-amphetamine are shown in FIG. 17. Pharmacokinetic parametersfor 1N administration of L-lysine-d-amphetamine are summarized in Table20.

TABLE 20 Pharmacokinetic parameters of d-amphetamine vs.L-lysine-d-amphetamine hydrochloride by IN administration AUC (0-1.5 h)Percent C_(max) Percent Drug ng · h/ml d-amphetamine (ng/ml)d-amphetamine Amphetamine 727 100 1,377 100 L-lysine-d- 4 0.5 7 0.5amphetamine

The process of part a was repeated using L-lysine-d-amphetamine mesylatesalt:

TABLE 21 Pharmacokinetic parameters of d-amphetamine vs.L-lysine-d-amphetamine mesylate salt by IN administration AUC (0-1.0 h)Percent C_(max) Percent Drug ng · h/ml d-amphetamine (ng/ml)d-amphetamine Amphetamine 573 100 1114 100 L-lysine-d- 25 4 26 2amphetamine mesylate salt

This example illustrates that when lysine is conjugated to the activeagent d-amphetamine, the bioavailability by the intranasal route issubstantially decreased, thereby diminishing the ability to abuse thedrug by this route.

Example 12 Intravenous Bioavailability of Amphetamine vs.L-lysine-d-amphetamine Dimesylate

Male Sprague-Dawley rats were dosed by intravenous tail vein injectionwith 1.5 mg/kg of d-amphetamine or L-lysine-d-amphetamine containing theequivalent amount of amphetamine. As observed with IN dosing, theconjugate did not release a significant amount of d-amphetamine. Mean(n=4) plasma concentration curves of amphetamine vs.L-lysine-d-amphetamine are shown in FIG. 19. Pharmacokinetic parametersfor IV administration of L-lysine-d-amphetamine are summarized in Table22.

TABLE 22 Pharmacokinetic parameters of d-amphetamine vs.L-lysine-d-amphetamine by IV administration AUC (0-1.5 h) PercentC_(max) Percent Drug ng · h/ml Amphetamine (ng/ml) AmphetamineAmphetamine 190 100 169 100 K-amphetamine 6 3 5 3

This example illustrates that when lysine is conjugated to the activeagent amphetamine, the bioavailability of amphetamine by the intravenousroute is substantially decreased, thereby diminishing the ability toabuse the drug by this route.

Example 13 Oral Bioavailability of L-lysine-d-amphetamine DimesylateCompared to d-amphetamine at Escalating Doses

The fraction of intact L-lysine-d-amphetamine absorbed following oraladministration in rats increased non-linearly in proportion toescalating doses from 1.5 to 12 mg/kg (FIG. 21-FIG. 25). The fractionabsorbed at 1.5 mg/kg was only 2.6 percent whereas it increased to 24.6percent by 12 mg/kg. The fraction absorbed fell to 9.3 percent at thehigh dose of 60 mg/kg. T_(max) ranged from 0.25 to 3 hours, and peakconcentrations occurred earlier for L-lysine-d-amphetamine than ford-amphetamine. L-lysine-d-amphetamine was cleared more rapidly thand-amphetamine with nearly undetectable concentrations by 8 hours at thelowest dose.

The bioavailability (AUC) of d-amphetamine from each drug administeredwas approximately equivalent at low doses. T_(max) for d-amphetaminefrom L-lysine-d-amphetamine ranged from 1.5 to 5 hours as compared to0.5 to 1.5 following administration of d-amphetamine sulfate. Thedifference in T_(max) was greater at higher doses. C_(max) ofd-amphetamine from L-lysine-d-amphetamine was reduced by approximatelyhalf as compared to the C_(max) of d-amphetamine from d-amphetaminesulfate administration at doses of 1.5 to 6 mg/kg, doses approximatingtherapeutic human equivalent doses (HEDs). Thus, at therapeutic doses,the pharmacokinetics of d-amphetamine from L-lysine-d-amphetamineresembled that of a sustained release formulation.

HEDs are defined as the equivalent dose for a 60 kg person in accordanceto the body surface area of the animal model. The adjustment factor forrats is 6.2. The HED for a rat dose of 1.5 mg/kg of d-amphetamine, forexample, is equivalent to 1.5/6.2×60=14.52 d-amphetamine base; which isequivalent to 14.52/0.7284=19.9 mg d-amphetamine sulfate, when adjustedfor the salt content.

TABLE 23 Human Equivalent Doses (HEDs) of d-amphetamine sulfate Rat doseof d-amphetamine Human equivalent dose (HED) of (mg/kg) d-amphetaminesulfate (mg) 1.5 19.9 3 39.9 6 79.7 12 159.4 30 399 60 797.2

At suprapharmacological doses (12 and 60 mg/kg), C_(max) was reduced by73 and 84 percent, respectively, as compared to d-amphetamine sulfate.For these high doses, the AUCs for d-amphetamine fromL-lysine-d-amphetamine were substantially decreased compared to those ofd-amphetamine sulfate, with the AUC_(inf) reduced by 76% at the highestdose (60 mg/kg). At 60 mg/kg, the levels of d-amphetamine fromd-amphetamine sulfate spiked rapidly; the experimental time course couldnot be completed due to extreme hyperactivity necessitating humaneeuthanasia.

In summary, oral bioavailability of d-amphetamine fromL-lysine-d-amphetamine decreased to some degree at higher doses.However, pharmacokinetics with respect to dose were nearly linear forL-lysine-d-amphetamine at doses from 1.5 to 60 mg/kg with the fractionabsorbed ranging from 52 to 81 percent (extrapolated from 1.5 mg/kgdose). Pharmacokinetics of d-amphetamine sulfate was also nearly linearat lower doses of 1.5 to 6 mg/kg with the fraction absorbed ranging from62 to 84 percent. In contrast to L-lysine-d-amphetamine, however,parameters were disproportionately increased at higher doses ford-amphetamine sulfate with the fraction absorbed calculated as 101 and223 percent (extrapolated from 1.5 mg/kg dose), respectively, for thesuprapharmacological doses of 12 and 60 mg/kg.

The results suggest that the capacity for clearance of d-amphetaminewhen delivered as the sulfate salt becomes saturated at the higher doseswhereas the gradual hydrolysis of L-lysine-d-amphetamine precludessaturation of d-amphetamine elimination at higher doses. The differencein proportionality of dose to bioavailability (C_(max) and AUC) ford-amphetamine and L-lysine-d-amphetamine is illustrated in FIG. 26-FIG.28. The pharmacokinetic properties of L-lysine-d-amphetamine as comparedto d-amphetamine at the higher doses decrease the ability to escalatedoses. This improves the safety and reduces the abuse liability ofL-lysine-d-amphetamine as a method of delivering d-amphetamine for thetreatment of ADHD or other indicated conditions.

Example 14 Intranasal Bioavailability of L-lysine-d-amphetamineDimesylate Compared to d-amphetamine

As shown in FIG. 31 and FIG. 32, bioavailability of d-amphetaminefollowing bolus intranasal administration of L-lysine-d-amphetamine wasapproximately 5 percent of that of the equivalent d-amphetamine sulfatedose with AUC_(inf) values of 56 and 1032, respectively. C_(max) ofd-amphetamine following L-lysine-d-amphetamine administration by theintranasal route was also about 5 percent of that of the equivalentamount of d-amphetamine sulfate with values of 78.6 ng/mL and 1962.9ng/mL, respectively. T_(max) of d-amphetamine concentration was delayedsubstantially for L-lysine-d-amphetamine (60 minutes) as compared toT_(max) of d-amphetamine sulfate (5 minutes), reflecting the gradualhydrolysis of L-lysine-d-amphetamine. Also, a high concentration ofintact L-lysine-d-amphetamine was detected following intranasaladministration. These results suggest that intranasal administration ofL-lysine-d-amphetamine provides only minimal hydrolysis ofL-lysine-d-amphetamine and thus only minimal release of d-amphetamine.

Example 15 Intravenous Bioavailability of L-lysine-d-amphetamineDimesylate Compared to d-amphetamine

As shown in FIG. 33 and FIG. 34, bioavailability of d-amphetaminefollowing bolus intravenous administration of L-lysine-d-amphetamine wasapproximately one-half that of the equivalent d-amphetamine sulfate dosewith AUC_(inf) values of 237.8 and 420.2, respectively. C_(max) ofd-amphetamine following L-lysine-d-amphetamine administration was onlyabout one-fourth that of the equivalent amount of d-amphetamine withvalues of 99.5 and 420.2, respectively. T_(max) of d-amphetamineconcentration was delayed substantially for L-lysine-d-amphetamine (30minutes) as compared to T_(max) of d-amphetamine sulfate (5 minutes),reflecting the gradual hydrolysis of L-lysine-d-amphetamine. Inconclusion, the bioavailability of d-amphetamine by the intravenousroute is substantially decreased and delayed when given asL-lysine-d-amphetamine. Moreover, bioavailability is less than thatobtained by oral administration of the equivalent dose ofL-lysine-d-amphetamine.

Summary of LC/MS/MS Bioavailability Data in Rats

The following tables summarize the bioavailability data collected in theexperiments discussed in Examples 13-15. Table 24, Table 25, and Table26 summarize the pharmacokinetic parameters of d-amphetamine followingoral, intranasal, and intravenous administration, respectively, ofd-amphetamine or L-lysine-d-amphetamine.

TABLE 24 Pharmacokinetic parameters of d-amphetamine following oraladministration of L-lysine-d-amphetamine or d-amphetamine at escalatingdoses AUC_(inf) Dose C_(max) T_(max) AUC₀₋₈ AUC_(inf) alt. F AUC/DoseC_(max)/Dose Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL) calc.* (%)(ng · h · kg/mL/mg) (ng · kg/mL/mg) K-Amp 1.5 59.6 3   308 331 376 61220.7 39.7 Amp 1.5 142.2 0.5   446 461 483 84 307.3 94.8 K-Amp 3 126.91.5   721 784 963 72 261.3 42.3 Amp 3 217.2 1.5   885 921 1,059 84 307.072.4 K-Amp 6 310.8 3 1,680 1,797 2,009 82 299.5 51.8 Amp 6 815.3 0.251,319 1,362 1429 62 227.0 135.9 K-Amp 12 412.6 5 2,426 2,701 2,701 62225.1 34.4 Amp 12 1,533.1 0.25 4,252 4,428 4,636 101 369.0 127.8 K-Amp60 2,164.3 5   9995.1 11,478 11,478 52 191.3 36.1 Amp 60 13,735 1 14,281** 48,707 48,707 223 811.8 228.9 *An alternative calculation ofAUC_(inf) can be performed using WinNonlin ® software (Version 4.1,Pharsight, Inc., Mountain View, California). **AUC (0-1.5)

TABLE 25 Pharmacokinetic parameters of d-amphetamine following bolusintravenous administration of L-lysine-d-amphetamine (1.5 mg/kg) DoseC_(max) T_(max) AUC₀₋₂₄ AUC₀₋₂₄ AUC_(inf) AUC_(inf) Route Drug (mg/kg)(ng/mL) (h) (ng · h/mL) alt. calc.* (ng · h/mL) alt. calc.* IV K-Amp 1.599.5 0.5 237.8 207 237.9 218 IV Amp 1.5 420.2 0.083 546.7 511 546.9 521

TABLE 26 Pharmacokinetic parameters of d-amphetamine followingintranasal administration of L-lysine-d-amphetamine Dose AUC₀₋₁AUC_(inf) AUC_(inf) Route Drug (mg/kg) C_(max) (ng/mL) T_(max) (h) (ng ·h/mL) (ng · h/mL) alt. calc.* IN K-Amp 3 78.6 1 56 91 NA IN Amp 3 1962.90.083 1032 7291 1,267

Table 27, Table 28, and Table 29 summarize the pharmacokineticparameters of L-lysine-d-amphetamine following oral, intravenous, andintranasal administration of L-lysine-d-amphetamine.

TABLE 27 Pharmacokinetic parameters of L-lysine-d-amphetamine followingoral administration of L-lysine-d-amphetamine at escalating doses DoseC_(max) T_(max) AUC₀₋₈ AUC_(inf) AUC_(inf) F Route Drug (mg/kg) (ng/ml)(ng/ml) (ng · h/mL) (ng · h/mL) alt. calc.* (%) Oral K-Amp 1.5 36.5 0.2559.4 60 60 2.6 Oral K-Amp 3 135.4 1.5 329.7 332.1 331 7.2 Oral K-Amp 6676.8 0.25 1156.8 1170.8 1,176 12.8 Oral K-Amp 12 855.9 1 4238.6 4510.45,169 24.6 Oral K-Amp 60 1870.3 3 8234.3 8499.9 8,460 9.3

TABLE 28 Pharmacokinetic parameters of L-lysine-d-amphetamine followingbolus intravenous administration of L-lysine-d-amphetamine Dose C_(max)T_(max) AUC₀₋₂₄ AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng· h/mL) IV K-Amp 1.5 4513.1 0.083 2,282 2,293

TABLE 29 Pharmacokinetic parameters of L-lysine-d-amphetamine followingintranasal administration of L-lysine-d-amphetamine Dose C_(max) T_(max)AUC₀₋₁ AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL)IN K-Amp 3 3345.1 0.25 2,580 9,139

Table 30 and Table 31 summarize the percent bioavailability ofd-amphetamine following oral, intranasal, and intravenous administrationof L-lysine-d-amphetamine as compared to d-amphetamine sulfate.

TABLE 30 Percent bioavailability (AUC_(inf)) of d-amphetamine followingadministration of L-lysine-d-amphetamine by various routes as comparedto bioavailability following administration of d-amphetamine sulfateDose (mg/kg) d-amphetamine base 1.5 3 6 12 60 HED 19.9 39.9 79.7 159.4797.2 Oral 72 85 132 61 24 IV 43 NA NA NA NA IN NA 1 NA NA NA

TABLE 31 Percent bioavailability (C_(max)) of d-amphetamine followingadministration of L-lysine-d-amphetamine by various routes as comparedto bioavailability following administration of d-amphetamine sulfateDose (mg/kg) d-amphetamine base 1.5 3 6 12 60 HED 19.9 39.9 79.7 159.4797.2 Oral 42 58 38 27 16 IV 24 NA NA NA NA IN NA 4 NA NA NA

Table 32-Table 37 summarize the time-course concentrations ofd-amphetamine and L-lysine-d-amphetamine following oral, intranasal, andintravenous administration of d-amphetamine or L-lysine-d-amphetamine.

TABLE 32 Time-course concentrations of d-amphetamine following bolusintravenous administration of L-lysine-d-amphetamine or d-amphetaminesulfate (1.5 mg/kg) Concentration Time (ng/ml) (hours) K-Amp Amp sulfate0 0 0 0.083 52.8 420.2 0.5 99.5 249.5 1.5 47.1 97.9 3 21.0 38.3 5 9.013.2 8 3.7 4.3 24 0.1 0.2

TABLE 33 Time-course concentrations of L-lysine-d-amphetamine followingbolus intravenous administration of L-lysine-d- amphetamine (1.5 mg/kg)K-Amp Time concentration (hours) (ng/ml) 0 0 0.083 4513.1 0.5 1038.7 1.5131.4 3 19.3 5 17.9 8 8.7 24 11.5

TABLE 34 Time-course concentrations of d-amphetamine following oraladministration of L-lysine-d-amphetamine at various doses TimeConcentration (ng/ml) (hours) 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 60mg/kg 0 0 0 0 0 0 0.25 20.5 25.3 96 54.3 90.9 0.5 34 40.9 140.2 96 175.11 46.7 95.1 225.9 233.3 418.8 1.5 40.7 126.9 268.4 266 440.7 3 59.6 105310.8 356.8 1145.5 5 38.6 107.6 219.5 412.6 2164.3 8 17.1 48 86 225.11227.5

TABLE 35 Time-course concentrations of d-amphetamine following oraladministration of d-amphetamine sulfate at various doses TimeConcentration (ng/ml) (hours) 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 60mg/kg 0 0 0 0 0 0 0.25 107.1 152.6 815.3 1533.1 6243.6 0.5 142.2 198.4462.7 1216 7931.6 1 105.7 191.3 301.3 828.8 13735.2 1.5 129.5 217.2 314904.8 11514.9 3 52.6 135.3 134.6 519.9 NA 5 29.5 73.5 77.4 404.3 NA 811.5 25.7 31.8 115.4 NA

TABLE 36 Time-course concentrations of d-amphetamine followingintranasal administration of L-lysine-d-amphetamine or d-amphetaminesulfate (3 mg/kg) Concentration Time (ng/ml) (hours) K-Amp Amp sulfate 00 0 0.083 31.2 1962.9 0.25 45.3 1497.3 0.5 61.3 996.2 1 78.6 404.6 AUC56 1032.3

TABLE 37 Time-course concentrations of L-lysine-d-amphetamine followingintranasal administration of L-lysine-d- amphetamine (3 mg/kg)Concentration Time (ng/ml) (hours) K-Amp 0 0 0.083 3345.1 0.25 3369.70.5 2985.8 1 1359.3

Example 16 Bioavailability of L-lysine-d-amphetamine Dimesylate ord-amphetamine Sulfate in Dogs (LC/MS/MS Analysis)

Example Experimental Design:

This was a non-randomized, two-treatment crossover study. All animalswere maintained on their normal diet and were fasted overnight prior toeach dose administration. L-lysine-d-amphetamine dose was based on thebody weight measured on the morning of each dosing day. The actual dosedelivered was based on syringe weight before and after dosing. Serialblood samples were obtained from each animal by direct venipuncture of ajugular vein using vacutainer tubes containing sodium heparin as theanticoagulant. Derived plasma samples were stored frozen until shipmentto Quest Pharmaceutical Services, Inc. (Newark, Del.). Pharmacokineticanalysis of the plasma assay results was conducted by Calvert. Animalswere treated as follows:

Number Dose Dose of Dogs/ Route of Conc. Dose Vol. Level SexAdministration Treatment (mg/mL) (mL/kg) (mg/kg) 3/M PO 1 0.2 10 1 3/MIV 2 1 2 1

Administration of the Test Article:

Oral: The test article was administered to each animal via a single oralgavage. On Day 1, animals received the oral dose by gavage using anesophageal tube attached to a syringe. Dosing tubes were flushed withapproximately 20 mL tap water to ensure the required dosing solution wasdelivered.

Intravenous: On Day 8, animals received L-lysine-d-amphetamine as asingle 30-minute intravenous infusion into a cephalic vein.

Sample Collection:

Dosing Formulations: Post-dosing, remaining dosing formulation was savedand stored frozen.

Blood: Serial blood samples (2 mL) were collected using venipuncturetubes containing sodium heparin. Blood samples were taken at 0, 0.25,0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours post-oral dosing. Bloodsamples were collected at 0, 0.167, 0.33, 0.49 (prior to stop ofinfusion), 0.583, 0.667, 0.75, 1, 2, 3, 4, 8, 12, and 23 hourspost-intravenous infusion start. Collected blood samples were chilledimmediately.

Plasma: Plasma samples were obtained by centrifugation of blood samples.Duplicate plasma samples (about 0.2 mL each) were transferred intoprelabeled plastic vials and stored frozen at approximately −70° C.

Sample Assay:

Plasma samples were analyzed for L-lysine-d-amphetamine andd-amphetamine using a validated LC-MS/MS method with an LLOQ of 1 ng/mLfor both analytes.

Microsoft Excel (Version 6, Microsoft Corp., Redmond, Wash.) was usedfor calculation of mean plasma concentration and graphing of the plasmaconcentration-time data. Pharmacokinetic analysis (non-compartmental)was performed using the WinNonlin® software program (Version 4.1,Pharsight, Inc. Mountain View, Calif.). The maximum concentration(C_(max)) and the time to C_(max)(T_(max)) were observed values. Thearea under the plasma concentration-time curve (AUC) was determinedusing linear-log trapezoidal rules. The apparent terminal rate constant(λz) was derived using linear least-squares regression with visualinspection of the data to determine the appropriate number of points(minimum of 3 data points) for calculating λz. The AUC_(0-inf) wascalculated as the sum of AUC_(0-t) and Cpred/λz, where Cpred was thepredicted concentration at the time of the last quantifiableconcentration. The plasma clearance (CL/F) was determined as the ratioof Dose/AUC_(0-inf). The mean residence time (MRT) was calculated as theratio of AUMC_(0-inf)/AUC_(0-inf) where AUMC_(0-inf) was the area underthe first moment curve from the time zero to infinity. The volume ofdistribution at steady state (V_(ss)) was estimated as CL*MRT. Half-lifewas calculated as ln 2/λz. The oral bioavailability (F) was calculatedas the ratio of AUC_(0-inf) following oral dosing to AUC_(0-inf)following intravenous dosing. Descriptive statistics (mean and standarddeviation) of the pharmacokinetic parameters were calculated usingMicrosoft Excel.

The objectives of this study were to characterize the pharmacokineticsof L-lysine-d-amphetamine and d-amphetamine following administration ofL-lysine-d-amphetamine in male beagle dogs. As shown in FIG. 35, in across-over design, L-lysine-d-amphetamine was administered to 3 malebeagle dogs orally and intravenously. Blood samples were collected up to24 and 72 hours after the intravenous and oral doses, respectively.

The mean L-lysine-d-amphetamine and d-amphetamine plasmaconcentration-time profiles following an intravenous or oral dose ofL-lysine-d-amphetamine are presented in FIG. 37 and FIG. 38,respectively. Comparative profiles of L-lysine-d-amphetamine tod-amphetamine following both routes are depicted in FIG. 35 and FIG. 36.Individual plots are depicted in FIG. 39 and FIG. 40. Thepharmacokinetic parameters are summarized in Table 38-Table 46.

Following a 30-minute intravenous infusion of L-lysine-d-amphetamine,the plasma concentration reached a peak at the end of the infusion.Post-infusion L-lysine-d-amphetamine concentration declined very rapidlyin a biexponential manner, and fell below the quantifiable limit (1ng/mL) by approximately 8 hours post-dose. Results of non-compartmentalpharmacokinetic analysis indicate that L-lysine-d-amphetamine is a highclearance compound with a moderate volume of distribution (V_(ss))approximating total body water (0.7 L/kg). The mean clearance value was2087 mL/h·kg (34.8 mL/min·kg) and was similar to the hepatic blood flowin the dog (40 mL/min·kg).

L-lysine-d-amphetamine was rapidly absorbed after oral administrationwith T_(max) at 0.5 hours in all three dogs. Mean absolute oralbioavailability was 33%, which suggests that L-lysine-d-amphetamine isvery well absorbed in the dog. The apparent terminal half-life was 0.39hours, indicating rapid elimination, as observed following intravenousadministration.

Plasma concentration-time profiles of d-amphetamine followingintravenous or oral administration of L-lysine-d-amphetamine weresimilar. See Table 39. At a 1 mg/kg oral dose of L-lysine-d-amphetamine,the mean C_(max) of d-amphetamine was 104.3 ng/mL. The half-life ofd-amphetamine was 3.1 to 3.5 hours, much longer when compared toL-lysine-d-amphetamine.

In this study, L-lysine-d-amphetamine was infused over a 30 minute timeperiod. Due to rapid clearance of L-lysine-d-amphetamine it is likelythat bioavailability of d-amphetamine from L-lysine-d-amphetamine woulddecrease if a similar dose were given by intravenous bolus injection.Even when given as an infusion the bioavailability of d-amphetamine fromL-lysine-d-amphetamine did not exceed that of a similar dose givenorally and the time to peak concentration was substantially delayed.This data further supports that L-lysine-d-amphetamine affords adecrease in the abuse liability of d-amphetamine by intravenousinjection.

TABLE 38 Pharmacokinetic parameters of L-lysine-d-amphetamine in malebeagle dogs following oral or intravenous administration ofL-lysine-d-amphetamine (1 mg/kg d-amphetamine base) Dose C_(max)AUC_(inf) CL/F V_(ss) Route mg/kg ng/mL T_(max) ^(a) h ng · h/mL t_(1/2)h MRT h mL/h · kg mL/kg F % IV 1 1650  0.49 964 0.88 0.33 2087 689 NA(0.00) (178) (0.49-0.49) (97.1) (0.2) (0.03)  (199) (105.9) Oral 1  328.2 0.5  319 0.39 0.81 6351 NA 33 (0.00)   (91.9) (0.5-0.5) (46.3)(0.1) (0.19)   (898.3) (1.9) ^(a)median (range) Abbreviations ofpharmacokinetic parameters are as follows: C_(max), maximum observedplasma concentration; T_(max), time when C_(max) observed; AUC_(0-t),total area under the plasma concentration versus time curve from 0 tothe last data point; AUC_(0-inf), total area under the plasmaconcentration versus time curve; t_(1/2), apparent terminal half-life;MRT, mean residence time; CL/F, oral clearance; V_(ss), volume ofdistribution at steady state; F, bioavailability.

TABLE 39 Pharmacokinetic parameters of d-amphetamine in male beagle dogsfollowing oral or intravenous administration of L-lysine-d-amphetamine(1 mg/kg d-amphetamine base) Dose C_(max) T_(max) ^(a) AUC_(inf) t_(1/2)Route (mg/kg) (ng/mL) (h) (ng · h/mL) (h) IV 1 113.2 1.0 672.5 3.14(0.00) (3.2) (0.67-2.0)  (85.7) (0.4) Oral 1 104.3 2.0 728.0 3.48 (0.00)(21.8) (2-2) (204.9) (0.4) ^(a)median (range)

TABLE 40 Pharmacokinetics of L-lysine-d-amphetamine in male beagle dogsfollowing 30 min intravenous administration of L-lysine-d-amphetamine (1mg/kg d-amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf)t_(1/2) CL V_(ss) MRT Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h)(mL/h/kg) (mL/kg) (h) 1 1470.3 0.49 898.2 900.2 0.72 2222 807.4 0.36 21826.4 0.49 1072.3 1076.1 ND^(b) 1859 603.4 0.32 3 1654.2 0.49 914.1916.9 1.05 2181 656.0 0.30 Mean 1650 0.49 961.5 964.4 0.88 2087 689.00.33 SD 178 0.49-0.49 96.0 97.1 0.2 199 105.9 0.03 ^(a)median (range);^(b)not determined CL, clearance following IV administration

TABLE 41 Pharmacokinetic parameters of L-lysine-d-amphetamine in malebeagle dogs following oral administration of L-lysine-d-amphetamine (1mg/kg d-amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf)t_(1/2) CL/F MRT F Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h)(mL/h/kg) (h) (%) 1 350.2 0.5 275.3 277.1 0.24 7218 0.68 30.8 2 407.20.5 367.8 368.7 0.48 5424 0.74 34.3 3 227.4 0.5 310.8 312.0 0.45 64101.03 34.0 Mean 328.2 0.5 318.0 319.3 0.39 6351 0.81 33.0 SD 91.9 0.046.7 46.3 0.1 898.3 0.19 1.9 ^(a)median (range)

TABLE 42 Pharmacokinetics of d-amphetamine in male beagle dogs following30 min. intravenous administration of L-lysine-d-amphetamine (1 mg/kgd-amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) DogID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h) 1 111.2 2.0 751.9 757.6 3.352 116.8 0.67 668.5 673.7 3.43 3 111.4 1.0 557.8 586.1 2.65 Mean 113.21.00 659.4 672.5 3.14 SD 3.2 0.67-2.0 97 85.7 0.4 ^(a)median (range)

TABLE 43 Pharmacokinetics of d-amphetamine in male beagle dogs followingoral administration of L-lysine-d-amphetamine (1 mg/kg d-amphetaminebase) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) Dog ID (ng/mL)(h) (ng · h/mL) (ng · h/mL) (h) 1 102.1 2.0 686.34 696.89 3.93 2 127.22.0 937.57 946.62 3.44 3 83.7 2.0 494.61 540.38 3.06 Mean 104.3 2.0706.2 728.0 3.48 SD 21.8 2.0-2.0 222.1 204.9 0.4 ^(a)median (range)

TABLE 44 Pharmacokinetics of d-amphetamine in male beagle dogs followingoral administration of L-lysine-d-amphetamine or d-amphetamine sulfate(1.8 mg/kg d-amphetamine base) Coefficient Mean Plasma Standard ofVariation Time Concentration Deviation (SD) (CV) (hours) Amp K-Amp AmpK-Amp Amp K-Amp 0 0 0 0 0 0 0 1 431.4 223.7 140.7 95.9 32.6 42.9 2 360291.8 87.6 93.6 24.3 32.1 4 277.7 247.5 68.1 66 24.5 26.7 6 224.1 214.759.3 62.1 26.5 28.9 8 175.4 150 66.7 40.1 38.0 26.7 12 81.4 47.6 58.7 1972.1 39.9 16 33 19.6 28.1 9 85.2 45.9 24 7.2 4.5 4.5 1.7 62.5 37.8

TABLE 45 Pharmacokinetics of d-amphetamine in female beagle dogsfollowing oral administration of L-lysine-d-amphetamine or d-amphetaminesulfate (1.8 mg/kg d-amphetamine base) Coefficient Mean Plasma Standardof Variation Time Concentration Deviation (SD) (CV) (hours) Amp K-AmpAmp K-Amp Amp K-Amp 0 0 0 0 0 0 0 1 217.8 308.8 141.7 40.7 65.1 13.2 2273.5 308 113.7 29.6 41.6 9.6 4 266 260.9 132.7 37.3 49.9 14.3 6 204.7212.1 84.5 38.7 41.3 18.2 8 160.1 164.3 72.7 43.5 45.4 26.5 12 79.4 68.741.3 31 52.0 45.1 16 25.5 22.3 13.4 4.7 52.5 21.1 24 5.6 5.4 4.1 1.973.2 35.2

TABLE 46 Pharmacokinetic parameters of d-amphetamine in male and femalebeagle dogs following oral administration of L-lysine-d-amphetamine ord-amphetamine sulfate (1.8 mg/kg d-amphetamine base) Males FemalesCompound Compound Parameter Amp K-Amp Amp K-Amp AUC_(inf) 3088.9 2382.22664.5 2569.9 Percent 100 77 100 96 C_(max) 431.4 291.8 308.8 273.5Percent 100 67 100 89 T_(max) (hours) 1 2 1 2 Percent 100 200 100 200

Example 17 Delayed Cardiovascular Effects of L-lysine-d-amphetamineDimesylate as Compared to d-amphetamine Following Intravenous Infusion

Systolic and diastolic blood pressure (BP) are increased byd-amphetamine even at therapeutic doses. Since L-lysine-d-amphetamine isexpected to release d-amphetamine (albeit slowly) as a result ofsystemic metabolism, a preliminary study was done using equimolar dosesof d-amphetamine or L-lysine-d-amphetamine to 4 dogs (2 male and 2female). The results suggest that the amide prodrug is inactive and thatslow release of some d-amphetamine, occurs beginning 20 minutes afterthe first dose. Relative to d-amphetamine, however, the effects are lessrobust. For example, the mean blood pressure is graphed in FIG. 43.Consistent with previously published data (Kohli and Goldberg, 1982),small doses of d-amphetamine were observed to have rapid effects onblood pressure. The lowest dose (0.202 mg/kg, equimolar to 0.5 mg/kg ofL-lysine-d-amphetamine) produced an acute doubling of the mean BPfollowed by a slow recovery over 30 minutes.

By contrast, L-lysine-d-amphetamine produced very little change in meanBP until approximately 30 minutes after injection. At that time,pressure increased by about 20-50%. Continuous release of d-amphetamineis probably responsible for the slow and steady increase in bloodpressure over the remaining course of the experiment. Upon subsequentinjections, d-amphetamine is seen to repeat its effect in a non-dosedependent fashion. That is, increasing dose 10-fold from the firstinjection produced a rise to the same maximum pressure. This may reflectthe state of catecholamine levels in nerve terminals upon successivestimulation of d-amphetamine bolus injections. Note that the rise inmean blood pressure seen after successive doses ofL-lysine-d-amphetamine (FIG. 43) produces a more gradual and lessintense effect. Similar results were observed for left ventricularpressure (FIG. 44). These results further substantiate the significantdecrease in d-amphetamine bioavailability by the intravenous route whengiven as L-lysine-d-amphetamine. As a result the rapid onset of thepharmacological effect of d-amphetamine that is sought by personsinjecting the drug is eliminated.

TABLE 47 Effects of L-lysine-d-amphetamine on cardiovascular parametersin the anesthetized dog (mean values, n = 2) % % % % Treatment Time SAPChange DAP Change MAP Change LVP Change 0.9% Saline 0 81 0 48 0 61 0 870 1 ml/kg 30 87 7 54 11 67 10 87 0 K-Amp 0 84 0 51 0 64 0 86 0 0.5 mg/kg5 87 4 52 3 66 3 87 2 15 93 11 51 1 67 5 95 11 25 104 25 55 8 73 15 10522 30 107 28 58 14 77 21 108 26 K-Amp 0 105 0 55 0 74 0 108 0 1.0 mg/kg5 121 15 63 15 85 15 120 11 15 142 35 73 33 100 35 140 29 25 163 55 9775 124 68 162 50 30 134 28 73 32 98 32 144 33 K-Amp 0 132 0 71 0 95 0144 0 5.0 mg/kg 5 142 7 71 0 99 4 151 5 15 176 33 98 39 130 37 184 28 25126 −5 69 −3 96 1 160 11 30 132 0 70 −1 99 4 163 13 SAP: systolicarterial pressure (mmHg); MAP: mean arterial pressure (mmHg); DAP:diastolic arterial pressure (mmHg); LVP: left ventricular pressure(mmHg); % Change: percent change from respective Time 0.

TABLE 48 Effects of d-amphetamine on cardiovascular parameters in theanesthetized dog (mean values, n = 2) % % % % Treatment Time SAP ChangeDAP Change MAP Change LVP Change 0.9% Saline 0 110 0 67 0 84 0 105 0 1ml/kg 30 108 −2 65 −3 82 −2 101 −3 d-amphetamine 0 111 0 67 0 84 0 104 00.202 mg/kg 5 218 97 145 117 176 109 214 107 15 168 52 97 45 125 49 15752 25 148 34 87 30 110 31 142 37 30 140 26 80 20 103 23 135 30d-amphetamine 0 139 0 78 0 101 0 133 0 0.404 mg/kg 5 240 73 147 88 18785 238 79 15 193 39 112 44 145 43 191 43 25 166 19 92 17 122 20 168 2630 160 16 87 11 117 16 163 22 d-amphetamine 0 158 0 87 0 115 0 162 02.02 mg/kg 5 228 44 128 48 169 47 227 40 15 196 24 107 23 142 23 200 2425 189 20 102 17 135 17 192 19 30 183 16 98 13 129 12 187 16

Example 18 Pharmacodynamic (Locomotor) Response to Amphetamine vs.L-lysine-d-amphetamine diHCl by Oral Administration

Male Sprague-Dawley rats were provided water ad libitum, fastedovernight, and dosed by oral gavage with 6 mg/kg of amphetamine orL-lysine-d-amphetamine containing the equivalent amount ofd-amphetamine. Horizontal locomotor activity (HLA) was recorded duringthe light cycle using photocell activity chambers (San DiegoInstruments). Total counts were recorded every 12 minutes for theduration of the test. Rats were monitored in three separate experimentsfor 5, 8, and 12 hours, respectively. Time vs. HLA counts ford-amphetamine vs. L-lysine-d-amphetamine is shown in FIG. 45 and FIG.46. In each experiment the time until peak activity was delayed, and thepharmacodynamic effect was evident for an extended period of time forL-lysine-d-amphetamine as compared to d-amphetamine. The total activitycounts for HLA of Lys-Amp dosed rats were increased (11-41%) over thoseinduced by d-amphetamine in all three experiments.

TABLE 49 Locomotor activity of rats orally administered d-amphetaminevs. L-lysine-d-amphetamine (5 h) Peak of activity Time of Time of TotalTotal Activity (Counts Peak Last Count Test Activity Counts Above per(Counts per Above 200 Material Counts Baseline 0.2 h) 0.2 h) per 0.2 hVehicle 4689 4174 80 1.4 — K-Amp 6417 5902 318 1.8   5 h Amp 515 0 2910.6 2.6 h

TABLE 50 Locomotor activity of rats orally administered d-amphetaminevs. L-lysine-d-amphetamine (12 h) Peak of activity Time of Time of TotalTotal Activity (Counts Peak Last Count Test Activity Counts Above per(Counts per Above 200 Material Counts Baseline 0.2 h) 0.2 h) per 0.2 hVehicle 936 0 81 7.2 — K-Amp 8423 7487 256 1.8 8.6 h Amp 6622 5686 2230.6 6.4 h

Example 19 Pharmacodynamic Response to d-amphetamine vs.L-lysine-d-amphetamine diHCl by Intranasal Administration

Male Sprague-Dawley rats were dosed by intranasal administration withd-amphetamine or L-lysine-d-amphetamine (1.0 mg/kg). In a second set ofsimilarly dosed animals, carboxymethyl cellulose (CMC) was added to thedrug solutions at a concentration of 62.6 mg/ml (approximately 2-foldhigher than the concentration of L-lysine-d-amphetamine and 5-foldhigher than the d-amphetamine content). The CMC drug mixtures weresuspended thoroughly before each dose was delivered. Locomotor activitywas monitored using the procedure described in Example 18. As shown inFIG. 47 and FIG. 48, the activity vs. time (1 hour or 2 hours) is shownfor amphetamine/CMC vs. L-lysine-d-amphetamine and compared to that ofamphetamine vs. L-lysine-d-amphetamine CMC. As seen in FIG. 47, additionof CMC to L-lysine-d-amphetamine decreased the activity response of INdosed rats to levels similar to the water/CMC control, whereas no effectwas seen on amphetamine activity by the addition of CMC. The increase inactivity over baseline of L-lysine-d-amphetamine with CMC was only 9%compared to 34% for L-lysine-d-amphetamine without CMC when compared toactivity observed for d-amphetamine dosed animals (Table 51). CMC had noobservable effect on d-amphetamine activity induced by INadministration.

TABLE 51 Locomotor activity of intranasal d-amphetamine vs.L-lysine-d-amphetamine with and without CMC Total Activity TotalActivity Counts Counts Drug n (1 h) Above Baseline Percent Amp Amp 3 858686 100 Amp CMC 3 829 657 100 K-Amp 4 408 237 35 K-Amp CMC 4 232 60 9Water 1 172 0 0 Water CMC 1 172 0 0

Example 20 Pharmacodynamic Response to d-amphetamine vs.L-lysine-d-amphetamine diHCl by Intravenous Administration

Male Sprague-Dawley rats were dosed by intravenous administration withd-amphetamine or L-lysine-d-amphetamine (1.0 mg/kg). The activityexpressed as total activity counts over a three hour period of time isshown in FIG. 49. The activity induced by L-lysine-d-amphetamine wassubstantially decreased, and time to peak activity was delayed. Theincrease in activity over baseline of L-lysine-d-amphetamine was 34% forL-lysine-d-amphetamine when compared to activity observed ford-amphetamine dosed animals (Table 52).

TABLE 52 Total activity counts after intravenous administration ofd-amphetamine vs. L-lysine-d-amphetamine Total Activity Counts Drug n (3h) Above Baseline Percent Amp Amp 3 1659 1355 100 K-Amp 4 767 463 34Water 1 304 0 0

Example 21 Decrease in Toxicity of Orally AdministeredL-lysine-d-amphetamine diHCl

Three male and three female Sprague Dawley rats per group were given asingle oral administration of L-lysine-d-amphetamine at 0.1, 1.0, 10,60, 100, or 1000 mg/kg (Table 53). Each animal was observed for signs oftoxicity and death on Days 1-7 (with Day 1 being the day of the dose),and one rat/sex/group was necropsied upon death (scheduled orunscheduled).

TABLE 53 Dosing chart for oral administration of L-lysine-d-amphetaminetoxicity testing No. of Animals Dose Concentration Groups M F TestArticle (mg/kg) (mg/mL) 1 3 3 L-lysine-d-amphetamine 0.1 0.01 2 3 3L-lysine-d-amphetamine 1.0 0.1 3 3 3 L-lysine-d-amphetamine 10 1.0 4 3 3L-lysine-d-amphetamine 60 6.0 5 3 3 L-lysine-d-amphetamine 100 10 6 3 3L-lysine-d-amphetamine 1000 100

Key observations of this study include:

-   -   All animals in Groups 1-3 showed no observable signs throughout        the conduct of the study.    -   All animals in Groups 4-6 exhibited increased motor activity        within two hours post-dose and which lasted into Day 2.    -   One female rat dosed at 1000 mg/kg was found dead on Day 2.        Necropsy revealed chromodacryorrhea, chromorhinorrhea, distended        stomach (gas), enlarged adrenal glands, and edematous and        distended intestines.    -   A total of 4 rats had skin lesions of varying degrees of        severity on Day 3.    -   One male rat dosed at 1000 mg/kg was euthanatized on Day 3 due        to open skin lesions on the ventral neck.    -   All remaining animals appeared normal from Day 4 through Day 7.

Animals were observed for signs of toxicity at 1, 2, and 4 h post-dose,and once daily for 7 days after dosing and cage-side observations wererecorded. Animals found dead, or sacrificed moribund were necropsied anddiscarded.

Cage-side observations and gross necropsy findings are summarized above.The oral LD50 of d-amphetamine sulfate is 96.8 mg/kg. ForL-lysine-d-amphetamine dimesylate, although the data are not sufficientto establish a lethal dose, the study indicates that the lethal oraldose of L-lysine-d-amphetamine is above 1000 mg/kg because only onedeath occurred out of a group of six animals. Although a second animalin this dose group was euthanatized on Day 3, it was done for humanereasons and it was felt that this animal would have fully recovered.Observations suggested drug-induced stress in Groups 4-6 that ischaracteristic of amphetamine toxicity (NTP, 1990; NIOSH REGISTRYNUMBER: SI1750000; Goodman et. al., 1985). All animals showed noabnormal signs on Days 4-7 suggesting full recovery at each treatmentlevel.

The lack of data to support an established lethal dose is believed to bedue to a putative protective effect of conjugating amphetamine withlysine. Intact L-lysine-d-amphetamine has been shown to be inactive, butbecomes active upon metabolism into the unconjugated form(d-amphetamine). Thus, at high doses, saturation of metabolism ofL-lysine-d-amphetamine into the unconjugated form may explain the lackof observed toxicity, which was expected at doses greater than 100mg/kg, which is consistent with d-amphetamine sulfate (NTP, 1990). Theformation rate of d-amphetamine and the extent of the formation ofamphetamine may both attribute to the reduced toxicity. Alternatively,oral absorption of L-lysine-d-amphetamine may also be saturated at suchhigh concentrations, which may suggest low toxicity due to limitedbioavailability of L-lysine-d-amphetamine.

Example 22 In Vitro Assessment of L-lysine-d-amphetamine diHClPharmacodynamic Activity

It was anticipated that the acylation of amphetamine, as in the aminoacid conjugates discussed here, would significantly reduce the stimulantactivity of the parent drug. For example, Marvola (1976) showed thatN-acetylation of amphetamine completely abolished the locomotor activityincreasing effects in mice. To confirm that the conjugate was notdirectly acting as a stimulant, we tested (NovaScreen, Hanover, Md.) thespecific binding of Lys-Amp (10⁻⁹ to 10⁻⁵ M) to human recombinantdopamine and norepinephrine transport binding sites using standardradioligand binding assays. The results (Table 54) indicate that theLys-Amp did not bind to these sites. It seems unlikely that theconjugate retains stimulant activity in light of these results. (MarvolaM. (1976) “Effect of acetylated derivatives of some sympathomimeticamines on the acute toxicity, locomotor activity and barbiturateanesthesia time in mice.” Acta Pharmacol Toxicol (Copenh) 38(5):474-89).

TABLE 54 Results from radioligand binding experiments withL-lysine-d-amphetamine Reference Ki (M) for Assay Radioligand CompoundRef. Cpd. Activity* NE Transporter [3H]-Nisoxetine Desipramine 4.1 ×10⁻⁹ No DA Transporter [3H]-WIN35428 GBR-12909 7.7 × 10⁻⁹ No *Noactivity is defined as producing between −20% and 20% inhibition ofradioligand binding (Novascreen).

TABLE 55 Percent inhibition of DAT and NET with L-lysine-d-amphetamineL-lysine-d-amphetamine (mol/L) % inhibition DAT % inhibition NET 10⁻⁹−10.46 8.15 10⁻⁷ 11.52 −11.75 10⁻⁵ −0.71 13.89

Example 23 In Vitro Assessment to Release Amphetamine fromL-lysine-d-amphetamine Dimesylate

“Kitchen tests” were performed in anticipation of attempts by illicitchemists to release free amphetamine from the amphetamine conjugate.Preferred amphetamine conjugates are resistant to such attempts. Initialkitchen tests assessed the amphetamine conjugates' resistance to water,acid (vinegar), and base (baking powder and baking soda) where in eachcase, the sample was heated to boiling for 20-60 minutes.L-lysine-d-amphetamine and GGG-Amp released no detectable freeamphetamine.

TABLE 56 In vitro assessment Vinegar Tap Water Baking Powder Baking SodaL-lysine-d- 0% 0% 0% 0% amphetamine Gly₃-Amp 0% 0% 0% 0%

Amphetamine conjugate stability was assessed under concentratedconditions, including concentrated HCl and in 10 N NaOH solution atelevated temperatures. Lys-Amp stock solutions were prepared in H₂O anddiluted 10-fold with concentrated HCl to a final concentration of 0.4mg/mL and a final volume of 1.5 mL. Samples were heated in a water bathto about 90° C. for 1 hour, cooled to 20° C., neutralized, and analyzedby HPLC for free d-amphetamine. The results suggest that only a minimalamount of d-amphetamine is released under these concentrated conditions.

TABLE 57 Stability under concentrated conditions % AUC solution Lys-Ampd-amphetamine 10 N NaOH 99 <1 conc. HCl 96 4

Amphetamine conjugate stability was assessed under acidic conditions.

TABLE 58 Acids used for stability study Acid Concentrations Hydrochloricacid 10%, 25%, 50%, 75%, and concentrated Acetic acid 10%, 25%, 50%,75%, and concentrated Sulfuric acid 10%, 25%, 50%, 75%, and concentratedPhosphoric acid 10%, 25%, 50%, 75%, and concentrated Nitric acid 10%,25%, 50%, 75%, and concentrated Citric acid 10%, 25%, 50%, 75%, andsaturated

At ambient temperature, only a limited amount of d-amphetamine wasreleased. At 90° C., only a limited amount of d-amphetamine wasreleased, but the decomposition of L-lysine-d-amphetamine was morepronounced. This suggested that the amide bond is stable, and that theconjugate usually degrades before an appreciable amount is hydrolyzed.At reflux conditions, concentrated hydrochloric acid and 50% sulfuricacid released 85% and 59%, respectively, of the d-amphetamine content,but rendered the drug in undesirable acidic solution. The process forrecovering d-amphetamine from the acidic solution further reduces theyield.

In a similar test, reflux in concentrated HCl resulted in somehydrolysis after 5 hours (28%) with further hydrolysis occurring after22 hours (76%). Reflux in concentrated H₂SO₄ for 2 hours resulted incomplete decomposition of Lys-Amp and potentially releasedd-amphetamine. As described above, recovery of d-amphetamine from theacidic solution would further reduce the yield.

Amphetamine conjugate stability was also assessed under basicconditions, including variable concentrations of sodium hydroxide,potassium hydroxide, sodium carbonate, ammonium hydroxide, diethylamine, and triethyl amine. The maximum d-amphetamine release was 25.4%obtained by 3M sodium hydroxide; all other basic conditions resulted ina release of less than 3%.

Example 24 Stability of L-lysine-d-amphetamine Dimesylate UnderTreatment with Commercially Available Products

The stability of L-lysine-d-amphetamine dimesylate was assessed undertreatment commercially available acids, bases, and enzyme cocktails. Foracids and bases (Table 59), 10 mg of Lys-Amp was mixed with 2 mL of eachstock solution, and the solution was shaken at 20° C. For enzymetreatment (Table 60), 10 mg Lys-Amp was mixed with 5 mL of each enzymecocktail, and the solution was shaken at 37° C. Each aliquot (0, 1, and24 h) was neutralized and filtered prior to analysis by HPLC. Many ofthe commercially available reagents also contained various solventsand/or surfactants.

Unless otherwise indicated, solutions were used directly from thecontainer and were combined with neat Lys-Amp solid. Lewis Red Devil®Lye, Enforcer Drain Care® Septic Treatment, and Rid-X® Septic Treatmentwere prepared as saturated solutions in H₂O. Enzymes used were purchasedfrom Sigma and directly dissolved in water (3 mg/mL pepsin, 10 mg/mLpancreatin, 3 mg/mL pronase, 3 mg/mL esterase), while enzyme-containingnutraceuticals such as Omnigest® and VitälZym® were first either crushedor opened (1 tablet or capsule per 5 mL of H₂O).

The commercial acids and bases were ineffective in hydrolyzing Lys-Amp.Only treatment with Miracle-Gro® (7% release) and Olympic® Deck Cleaner(4% release) showed any release, but even after 24 hours, the amount ofd-amphetamine was negligible. Among the enzyme products, only pureesterase (19% release) or pronase (24% release) mixtures successfullycleaved lysine (after 24 hours).

TABLE 59 Stability of L-lysine-d-amphetamine dimesylate under treatmentwith commercially available acids and bases d-amphetamine (1) (w.t. %d-amphetamine) Solution (active ingredients) 1 h 24 h Lysol ® Toilet(HCl) 0 0 Crete-nu (75% H₃PO₄) 0 0 CLR ® (sulfamic acid, hydroxyaceticacid) 0 0 Roebic ® Drain Flow (90% H₂SO₄) 0 0 Crown ® Muriatic Acid(31.45% HCl) 0 0 Liquid-Plumr ® (NaOH, NaClO, H₂O₂) 0 0 Brasso ® (NH₄OH)0 0 Johnson ® Wax Degreaser (K₂CO₃) 0 0 Miracle-Gro ® (Urea, K₃PO₄) 0 7Lewis Red Devil ® Lye (NaOH) 0 0 Drain Power (NaOH, NaClO) 0 0 SavogranTSP (Na₃PO₄) 0 0 Johnson ® Wax Stripper (NaOH) 0 0 Olympic ® DeckCleaner (NaOH, NaClO) 0 4 Windex ® (NH₄OH) 0 0 Greased Lightning ®(basic components) 0 0

TABLE 60 Stability of L-lysine-d-amphetamine dimesylate under treatmentwith commercially available enzyme cocktails d-amphetamine (1) (w.t. %d-amphetamine) Solution 1 h 24 h Cellfood ® 0 0 Drano ® Max withBacteria 0 0 VitälZym ® 0 0 Omnigest ® 0 0 Enforcer ® Septic 0 0 Rid-X ®Septic 0 0 Esterase 0 19 Pancreatin 0 0 Pepsin 0 0 Pronase 0 24

Example 25 Bioavailability of Various Peptide Amphetamine Conjugates(HCl Salts) Administered by Oral, Intranasal, and Intravenous Routes

Oral administration: Male Sprague-Dawley rats were provided water adlibitum, fasted overnight, and dosed by oral gavage with amphetamine oramino acid-amphetamine conjugates containing the equivalent amount ofamphetamine.

Intranasal administration: Male Sprague-Dawley rats were dosed byintranasal administration with amphetamine or lysine-amphetamine (1.8mg/kg).

The relative in vivo performance of various amino acid-amphetaminecompounds is shown in FIG. 50-FIG. 58 and summarized in Table 61.Intranasal bioavailability of amphetamine from Ser-Amp was decreased tosome degree relative to free amphetamine. However, this compound was notbioequivalent with amphetamine by the oral route of administration.Phenylalanine was bioequivalent with amphetamine by the oral route ofadministration, however, little or no decrease in bioavailability byparenteral routes of administration was observed. Gly₃-Amp had nearlyequal bioavailability (90%) by the oral route accompanied by a decreasein C_(max) (74%). Additionally, Gly₃-Amp showed a decrease inbioavailability relative to amphetamine by intranasal and intravenousroutes.

TABLE 61 Percent bioavailability of amino acid amphetamine compoundsadministered by oral, intranasal, or intravenous routes Oral IntranasalIntravenous Percent Percent Percent Percent Percent Percent Drug AUCC_(max) AUC C_(max) AUC C_(max) Amphetamine 100 100 100 100 100 100E-Amp 73 95 NA NA NA NA EE-Amp 26 74 NA NA NA NA EEE-Amp 69 53 10 10 NANA L-Amp 65 81 NA NA NA NA S-Amp 79/55 62/75 76 65 NA NA G-Amp 81 78 6553 NA NA GG-Amp 79 88 88 85 NA NA GGG-Amp 111/68  74/73 32 38 45 46F-Amp 95 91 97 95 87 89 EEF-Amp 42 73 39 29 NA NA FF-Amp 27 64 NA NA NANA Gulonate-Amp 1 1 0.4 0.5 3 5 K-Amp 98 55 0.5 0.5 3 3 KG-Amp 69 71 1312 NA NA dKlK-Amp 16 7 2 2 NA NA LE-Amp 40 28 6 6 NA NA H-Amp 16 21 2242 NA NA P-Amp 6 3 2 2 NA NA PP-Amp 61 80 47 43 NA NA Y-Amp 25 20 21 20NA NA I-Amp 71 52 73 97 NA NA

Several single amino acid amphetamine conjugates had comparable oralbioavailability (80-100%) to d-amphetamine. Lys, Gly, and Pheconjugates, for example, all demonstrated similar oral bioavailabilityto the parent drug. Dipeptide prodrugs generally showed lowerbioavailability than the respective amino acid analogs, and tripeptidecompounds displayed no discernable trend. Several amino acid amphetamineconjugates had decreased parenteral bioavailability. Preferredconjugates, such as Lys-Amp, exhibit both oral bioavailabilitycomparable to d-amphetamine and decreased parenteral bioavailabilitycompared to d-amphetamine.

Example 26 Decreased Oral C_(max) of d-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum, fastedovernight, and dosed by oral gavage with amphetamine conjugate ord-amphetamine sulfate. All doses contained equivalent amounts ofd-amphetamine base. Plasma d-amphetamine concentrations were measured byELISA (Amphetamine Ultra, 109319, Neogen, Corporation, Lexington, Ky.).The assay is specific for d-amphetamine with only minimal reactivity(0.6%) of the major d-amphetamine metabolite(para-hydroxy-d-amphetamine) occurring. Plasma d-amphetanine andL-lysine-d-amphetamine concentrations were measured by LC/MS/MS whereindicated in examples.

Example 27 Decreased Intranasal Bioavailability (AUC and C_(max)) ofd-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum and doses wereadministered by placing 0.02 ml of water containing amphetamineconjugate or d-amphetamine sulfate into the nasal flares. All dosescontained equivalent amounts of d-amphetamine base. Plasma d-amphetamineconcentrations were measured by ELISA (Amphetamine Ultra, 109319,Neogen, Corporation, Lexington, Ky.). The assay is specific ford-amphetamine with only minimal reactivity (0.6%) of the majord-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. Plasmad-amphetamine and L-lysine-d-amphetamine concentrations were measured byLC/MS/MS where indicated in examples.

Example 28 Decreased Intravenous Bioavailability (AUC and C_(max)) ofd-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum, and doses wereadministered by intravenous tail vein injection of 0.1 ml of watercontaining amphetamine conjugate or d-amphetamine sulfate. All dosescontained equivalent amounts of d-amphetamine base. Plasma d-amphetamineconcentrations were measured by ELISA (Amphetamine Ultra, 109319,Neogen, Corporation, Lexington, Ky.). The assay is specific ford-amphetamine with only minimal reactivity (0.6%) of the majord-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. Plasmad-amphetamine and L-lysine-d-amphetamine concentrations were measured byLC/MS/MS where indicated in examples.

Example 29 Attachment of Amphetamine to Variety of Chemical Moieties

The above examples demonstrate the use of an amphetamine conjugated to achemical moiety, such as an amino acid, which is useful in reducing thepotential for overdose while maintaining its therapeutic value. Theeffectiveness of binding amphetamine to a chemical moiety wasdemonstrated through the attachment of amphetamine to lysine (K),however, the above examples are meant to be illustrative only. Theattachment of amphetamine to any variety of chemical moieties (i.e.,peptides, glycopeptides, carbohydrates, nucleosides, or vitamins) asdescribed below through similar procedures using the following exemplarystarting materials.

Amphetamine Synthetic Examples:

Synthesis of Gly₂-Amp

-   -   Gly₂-Amp was synthesized by a similar method except the amino        acid starting material was Boc-Gly-Gly-OSu.

Synthesis of Glu₂-Phe-Amp

-   -   Glu2-Phe-Amp was synthesized by a similar method except the        amino acid starting material was Boc-Glu(OtBu)-Glu(OtBu)-OSu and        the starting drug conjugate was Phe-Amp (see Phe-Amp synthesis).

Synthesis of His-Amp

-   -   His-Amp was synthesized by a similar method except the amino        acid starting material was Boc-His(Trt)-OSu.

Synthesis of Lys-Gly-Amp

-   -   Lys-Gly-Amp was synthesized by a similar method except the amino        acid starting material was Boc-Lys(Boc)-OSu and the starting        drug conjugate was Gly-Amp (see Gly-Amp synthesis).

Synthesis of Lys-Glu-Amp

-   -   Lys-Glu-Amp was synthesized by a similar method except the amino        acid starting material was Boc-Lys(Boc)-OSu and the starting        drug conjugate was Glu-Amp.

Synthesis of Glu-Amp

-   -   Glu-Amp was synthesized by a similar method except the amino        acid starting material was Boc-Glu(OtBu)-OSu.

Synthesis of (d)-Lys-(l)-Lys-Amp

-   -   (d)-Lys-(l)-Lys-Amp was synthesized by a similar method except        the amino acid starting material was        Boc-(d)-Lys(Boc)-(l)-Lys(Boc)-OSu.

Synthesis of Gulonic Acid-Amp

-   -   Gul-Amp was synthesized by a similar method except the        carbohydrate starting material was gulonic acid-OSu.

Example 30 Lack of Detection of L-lysine-d-amphetamine diHCl in BrainTissue Following Oral Administration

Male Sprague-Dawley rats were provided water ad libitum, fastedovernight, and dosed by oral gavage with L-lysine-d-amphetamine ord-amphetamine sulfate. All doses contained equivalent amounts ofd-amphetamine base. As shown in FIG. 59, similar levels of d-amphetaminewere detected in serum as well as in brain tissue followingadministration of d-amphetamine sulfate or L-lysine-d-amphetamine. Thed-amphetamine from L-lysine-d-amphetamine showed a sustained presence inthe brain as compared to levels of d-amphetamine from d-amphetaminesulfate. The conjugate L-lysine-d-amphetamine was present in appreciableamounts in serum but was not detected in brain tissue indicating thatthe conjugate does not cross the blood brain barrier to access thecentral nervous system site of action.

Example 31 Pharmaceutical Composition of L-lysine-d-amphetamineDimesylate

A gelatin capsule dosage form was prepared in three dosage strengths.The hard gelatin capsules were printed with NRP104 and the dosagestrength. The capsule fill contains a white to off-white finely dividedpowder uniform in appearance.

TABLE 62 Composition of L-lysine-d-amphetamine dimesylate capsulesQuantity (mg) Ingredient 30 50 70 Placebo Function Grade L-lysine-d-30.0 50.0 70.0 0.0 Active amphetamine dimesylate Microcrystalline 15170.0 98.0 144.0 Filler/ NF (Avicel ® Cellulose diluent, PH-102)disintegrant Croscarmellose 4.69 3.12 4.37 3.75 Disintegrant NF SodiumMagnesium 1.88 1.88 2.63 2.25 Lubricant NF (5712) Stearate GelatinCapsule White/ White/ Med. White/ Carrier NF Size 3 Med. Lt. Orange/White Orange Blue Lt. Blue Total 187.5 125 175 150

Other diluents, disintegrants, lubricants, and colorants, etc. may beused. Also, a particular ingredient can be used to serve a differentfunction than those listed above.

The pharmaceutical composition was prepared by milling de-lumpedL-lysine-d-amphetamine dimesylate (size 20 mesh) with microcrystallinecellulose. The mixture was sieved through a 30 mesh screen and thenmixed with croscarmellose sodium. Pre-screened magnesium stearate (size30 mesh) was added, and the composition was mixed until uniform to formthe capsule fill.

Example 32 Clinical Pharmacokinetic Evaluation and Oral Bioavailabilityof L-lysine-d-amphetamine Dimesylate 70 mg Capsules Administered toHealthy Adults Under Fasting Conditions for 7 Days

In this open-label, single-arm study, healthy adults between the ages of18 to 55 years were administered 70 mg of L-lysine-d-amphetaminedimesylate with 8 ounces of water once daily (7 am) for 7 consecutivedays. Patients fasted for at least 10 hours before and 4 hours afterfinal dosing. Venous blood samples (7 mL) were drawn into EDTAvacutainers both before medication dosing on days 0, 1, 6, and 7 (in themorning) and at 16 time points (hours 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8,10, 12, 16, 24, 48, and 72) after final dosing on day 7. Immediatelyafter sample collection, vacutainer tubes were centrifuged at 3000 rpmat 4° C. for 10 minutes; within 1 hour of collection, they were storedat −20° C. Plasma samples were analyzed for L-lysine-d-amphetamine andd-amphetamine using a validated LC/MS/MS method.

By dose 5, d-amphetamine reached steady state. After dose 7, meanAUC₀₋₂₄ was 1113 ng·h/mL, mean AUC_(0-∞) was 1453 ng·h/mL, mean C_(max)was 90.1 ng·h/mL, and mean T_(max) was 3.68 hours. See Table 63 and FIG.60. In comparison, extended-release amphetamine salts exhibit a T_(max)of 5.8 hours and AUC_(0-∞) 853 ng·h/mL after an overnight fast. J. F.Auiler et al., “Effect of food on early drug exposure fromextended-release stimulants: results from the Concerta, Adderall XR FoodEvaluation (CAFE) study,” Curr Med Res Opin 18: 311-316 at 313 (2002).

Intact L-lysine-d-amphetamine was rapidly converted to d-amphetamine.After dose 7, mean AUC₀₋₂₄ was 60.66 ng·h/mL, and mean AUC_(0-∞) was61.06 ng·h/mL. See Table 63 and FIG. 60. In addition, mean C_(max) was47.9 ng·h/mL, and mean T_(max) was 1.14 hours for intactL-lysine-d-amphetamine. L-lysine-d-amphetamine was completely eliminatedwithin approximately 6 hours.

There were no gender differences in systemic exposure to d-amphetamine,though C_(max) was 12% higher in men after normalization by body weight.

The multidose pharmacokinetic profile of d-amphetamine released from theprodrug L-lysine-d-amphetamine is consistent with extended-releaseproperties. The adverse events that occurred in this setting areconsistent with other stimulants and suggest that suggest thatL-lysine-d-amphetamine 70 mg is well tolerated.

TABLE 63 Steady-state pharmacokinetics parameters (n = 11) ParameterMean SD CV % d-amphetamine C_(max) (ng/mL) 90.1 29.6 32.84 C_(min)(ng/mL) 18.2 14.2 78.12 T_(max) (h) 3.68 1.42 38.54 t_(1/2) (h) 10.082.76 27.37 AUC₀₋₂₄ (ng · h/mL) 1113 396.8 35.65 AUC_(0-∞) (ng · h/mL)1453 645.7 44.45 AUC_(0-t) (ng · h/mL) 1371 633.5 46.19 FI (%) 163.5537.20 22.74 Intact L-lysine-d-amphetamine C_(max) (ng/mL) 47.9 18.638.81 C_(min) (ng/mL) 0.0 0.0 — T_(max) (h) 1.14 0.32 28.45 t_(1/2) (h)0.43 0.09 21.90 AUC₀₋₂₄ (ng · h/mL) 60.66 21.00 34.61 AUC_(0-∞) (ng ·h/mL) 61.06 20.63 33.79 AUC_(0-t) (ng · h/mL) 59.44 21.47 36.12 FI (%)1896.06 340.24 17.94

Example 33 Clinical Pharmacokinetic Evaluation and Oral Bioavailabilityof L-lysine-d-amphetamine Dimesylate Compared to Amphetamine ExtendedRelease Products Adderall XR® and Dexedrine Spansule® Used in theTreatment of ADHD

TABLE 64 Treatment groups and dosage for clinical pharmacokineticevaluation of L-lysine-d-amphetamine compared to Adderall XR ® orDexedrine Spansule ® Dose Treatment No. of Dose (amphetamine Drug GroupSubjects Dose (mg) base) L-lysine- A 10 1 × 25 mg 25 7.37 d-amphetaminecapsule L-lysine- B 10 3 × 25 mg 75 22.1 d-amphetamine capsulesDexedrine C 10 3 × 10 mg 30 22.1 Spansule ® capsules Adderall XR ® D 101 × 30 mg 35 21.9 capsules plus 1 × 5 mg capsule

A clinical evaluation of the pharmacokinetics and oral bioavailabilityof L-lysine-d-amphetamine in humans was conducted.L-lysine-d-amphetamine was orally administered at doses approximatingthe lower (25 mg) and higher (75 mg) end of the therapeutic range basedon d-amphetamine base content of the doses. Additionally, the higherdose was compared to doses of Adderall XR® (Shire) or DexedrineSpansule® (GlaxoSmithKline) containing equivalent amphetamine base tothat of the higher L-lysine-d-amphetamine dose. Treatment groups anddoses are summarized in Table 64. All levels below limit quantifiable(blq<0.5 ng/mL) were treated as zero for purposes of pharmacokineticanalysis.

The concentrations of d-amphetamine and L-lysine-d-amphetamine intactconjugate following administration of L-lysine-d-amphetamine at the lowand high dose for each individual subject as well as pharmacokineticparameters are presented in Table 65-Table 70. The concentrations ofd-amphetamine following administration of Adderall XR® or DexedrineSpansule® for each individual subject as well as pharmacokineticparameters are presented in Table 69 and Table 70, respectively.Concentration-time curves showing L-lysine-d-amphetamine intactconjugate and d-amphetamine are presented in FIG. 61 and FIG. 62.Extended release of d-amphetamine from L-lysine-d-amphetamine wasobserved for both doses and pharmacokinetic parameters (C_(max) and AUC)were proportional to doses when the lower and higher dose results werecompared (FIG. 61 and FIG. 62). Significant levels of d-amphetamine werenot observed until one-hour post administration. Only small amounts (1.6and 2.0 percent of total drug absorption, respectively for 25 and 75 mgdoses; AUC_(inf)-molar basis) of L-lysine-d-amphetamine intact conjugatewere detected with levels peaking at about one hour (Table 66 and Table68). The small amount of intact conjugate absorbed was rapidly andcompletely eliminated, with no detectable concentrations present by fivehours, even at the highest dose.

In a cross-over design (identical subjects received Adderall XR® dosesfollowing a 7-day washout period), the higher L-lysine-d-amphetaminedose was compared to an equivalent dose of Adderall XR®. Adderall XR® isa once-daily extended release treatment for ADHD that contains a mixtureof d-amphetamine and l-amphetamine salts (equal amounts of d-amphetaminesulfate, d-/l-amphetamine sulfate, d-amphetamine saccharate, andd-/l-amphetamine aspartate). An equivalent dose of extended releaseDexedrine Spansule® (contains extended release formulation ofd-amphetamine sulfate) was also included in the study. As observed inpharmacokinetic studies in rats, oral administration ofL-lysine-d-amphetamine resulted in d-amphetamine concentration-timecurves similar to those of Adderall XR® and Dexedrine Spansule® (FIG. 63and FIG. 64). The bioavailability (AUC_(inf)) of d-amphetamine followingadministration of L-lysine-d-amphetamine was approximately equivalent toboth extended release amphetamine products (Table 71). Over the courseof twelve hours, typically the time needed for effective once-dailytreatment of ADHD, the bioavailability for L-lysine-d-amphetamine wasapproximately equivalent to that of Adderall XR® (d-amphetamine plusl-amphetamine levels) and over twenty percent higher than that ofDexedrine Spansule®. Based on the results of this clinical study,L-lysine-d-amphetamine would be an effective once-daily treatment forADHD. Moreover, L-lysine-d-amphetamine afforded similar pharmacokineticsin humans and animal models, namely, delayed release of d-amphetamineresulting in extended release kinetics. Based on these observationsL-lysine-d-amphetamine should also have abuse-resistant properties inhumans.

TABLE 65 Individual subject d-amphetamine concentrations andpharmacokinetic parameters following oral administration of a 25 mg doseof L-lysine-d-amphetamine to humans Subject Subject Subject SubjectSubject Subject Subject Subject Subject 102 103 105 107 110 112 113 116117 Subject 120 Mean SD CV % Time Hours 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 0 0 0.625 0 0 0 0 0.78 0.769 0 0.2 0.4 162.1 1 4.29 2.95 8.67 3.368.33 1.1 10 10.5 14 3.15 6.6 4.2 63.6   1.5 10 12.7 16 13.8 21.4 3.9424.7 19.5 24 15.1 16.1 6.5 40.3 2 16.3 18.4 17 21 25.9 9.29 30.9 23.6 3021.7 21.4 6.6 30.8 3 16.5 19.6 16.7 26.1 27 17.7 30.2 23.5 27.6 28.923.4 5.3 22.7 4 23.9 18.8 14.1 24.5 30.1 17.9 33.2 21.2 24.7 25.3 23.45.7 24.3 5 21.2 18.9 14.6 21.6 22.6 17.2 27 20 20.2 24.2 20.8 3.5 16.9 621.8 18 12.5 21.6 23.7 15.7 25.8 18.2 20.3 20.5 19.8 3.9 19.6 7 18.915.8 12.1 17.8 20.6 14.5 26.6 21 18.3 21.8 18.7 4.1 21.9 8 19.3 16.610.4 17.9 20 14.2 25.7 13.6 18.8 20.1 17.7 4.2 24.1 10  18.8 13.6 9.815.3 19.3 13.7 22.4 15.1 15.3 15.9 15.9 3.5 22.1 12  15.8 12.6 6.92 11.515.8 11.2 17.9 12 13.7 15.2 13.3 3.1 23.6 16  13.4 10.5 6.56 9.53 14.310.7 12.5 10.3 10 13 11.1 2.3 20.5 24  8.03 5.81 2.65 4.9 5.8 5.9 6.576.13 4.52 5.45 5.6 1.4 25.1 48  1.57 1.36 0 1.26 0.795 1.44 1.24 1.230.864 0.586 1.0 0.5 46.1 72  0 0 0 0 0 0 0 0 0 0 0 0 0 ParameterAUC_(0-12 h) 204.0 177.4 140.4 204.9 242.7 152.4 284.6 199.2 225.5 223.3205.4 42.5 20.7 (ng · h/mL) AUC_(last) (ng · h/mL) 463.3 375.1 201.4378.5 462.7 350.7 515.2 397.9 395.7 426.1 396.7 84.8 21.4 AUC_(inf) (ng· h/mL) 486.7 397.1 233.5 398.8 472 374 532.5 416.4 407 432.2 415.0 80.119.3 C_(max) (ng/mL) 23.9 19.6 17 26.1 30.1 17.9 33.2 23.6 30 28.9 25.05.6 22.3 T_(max) (hours) 4 3 2 3 4 4 4 2 2 3 3.1 0.876 28.2 T_(1/2)(hours) 10.32 11.18 8.36 11.18 8.16 11.22 9.68 10.43 9.06 7.22 9.68 1.4314.7

TABLE 66 Individual subject L-lysine-d-amphetamine intact conjugateconcentrations and pharmacokinetic parameters following oraladministration of a 25 mg dose of L-lysine-d-amphetamine to humansSubject Subject Subject Subject Subject Subject Subject CV 102 103 105107 110 112 113 Subject 116 Subject 117 Subject 120 Mean SD % Time Hours0 0 0 0 0 0 0 0 0 0 0 0 0 0   0.5 4.1 5.5 10.0 0.0 3.6 0.0 9.2 9.6 8.90.0 5.1 4.2 82.0 1 9.2 11.2 15.2 12.5 9.1 2.7 20.1 10.5 10.8 10.9 11.24.5 39.7   1.5 4.0 4.4 6.1 7.5 3.6 6.2 6.6 2.8 4.2 8.4 5.4 1.8 34.1 22.1 1.4 2.5 2.9 1.9 4.0 2.3 0 1.7 3.1 2.2 1.1 48.8 3 0 0 0 0 0 0 0 0 0 00 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 00 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 00 10  0 0 0 0 0 0 0 0 0 0 0 0 0 12  0 0 0 0 0 0 0 0 0 0 0 0 0 16  0 0 00 0 0 0 0 0 0 0 0 0 24  0 0 0 0 0 0 0 0 0 0 0 0 0 48  0 0 0 0 0 0 0 0 00 0 0 0 72  0 0 0 0 0 0 0 0 0 0 0 0 0 Parameter AUC_(last) 9.18 10.9516.31 10.68 8.583 5.439 18.51 10.77 12.35 10.41 11.32 3.74 33.1 (ng ·h/mL) AUC_(inf) 10.62 11.64 17.66 12.65 9.759 — 19.56 — 13.3 12.83 13.503.40 25.2 (ng · h/mL) C_(max) (ng/mL) 9.18 11.2 15.2 12.5 9.05 6.18 20.110.5 10.8 10.9 11.56 3.80 32.9 T_(max) (hours) 1 1 1 1 1 1.5 1 1 1 11.05 0.16 15.1 T_(1/2) (hours) 0.47 0.34 0.38 0.47 0.44 — 0.32 — 0.380.55 0.419 0.077 18.5

TABLE 67 Individual subject d-amphetamine concentrations andpharmacokinetic parameters following oral administration of a 75 mg doseof L-lysine-d-amphetamine to humans Subject Subject Subject SubjectSubject Subject Subject Subject Subject Subject 101 104 106 108 109 111114 115 118 119 Mean SD CV % Time Hours 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 0 0.748 0.506 0 0 0.779 0.525 0 3 1.85 0.7 1.0 132.2 1 11.9 14.412.6 7.26 5.9 10.3 7.2 23.1 23 27.9 14.4 7.7 53.6   1.5 40.3 34.6 30.422.8 19.3 38.4 19 52.8 51.5 55.8 36.5 13.8 37.8 2 84.6 48.9 68.2 34.832.7 57.2 33.1 91.3 61.7 70.4 58.3 21.0 36.0 3 72.9 64.3 55.7 60.3 62.361.1 44.8 95.8 62.1 83.6 66.3 14.5 21.9 4 84.6 65.3 58.8 51.1 77.9 63.347.6 89.2 54.2 86 67.8 15.5 22.8 5 65 55.6 60.2 74 83.9 59.1 56.9 77.754.9 82.8 67.0 11.5 17.2 6 71 53.5 49.4 51.5 78.3 50.8 55.1 68.8 52.9 6459.5 10.2 17.1 7 53.8 55.7 52.9 69.5 73.1 52.9 55.9 71.2 45.1 74.6 60.510.5 17.4 8 63.7 40.3 47.3 45.7 72.2 46.5 54.2 61.1 44.3 66.2 54.2 10.920.2 10  43.7 41.7 37 58.4 67 44.3 48.4 68 34.1 55.9 49.9 11.9 24.0 12 46.4 26.1 36.7 37.4 49.9 32.4 37.1 54.1 34.5 45.1 40.0 8.6 21.6 16  35.422.2 25.7 48 44.9 24.3 28.9 44.7 31.7 34.5 34.0 9.2 27.1 24  16.4 11.414.9 13.2 18.4 16.8 20.5 21.7 15.7 18.1 16.7 3.1 18.8 48  2.74 2.14 4.172.73 3.75 4.81 2.81 4.26 3.36 3.4 0.9 25.9 72  0 0 0 1.07 0.661 0.6871.49 0 0 0.553 0.4 0.5 120.2 Parameter AUC_(0-12 h) 666.2 525.9 531.6570.3 704.8 545.6 513.7 790.9 523.4 742.8 611.5 104.5 17.1 (ng · h/mL)AUC_(last) 1266 918.7 1031 1257 1442 1123 1223 1549 1143 1417 1237.0194.0 15.7 (ng · h/mL) AUC_(inf) 1301 948.3 1072 1278 1451 1133 12511582 1154 1425 1259.5 191.3 15.2 (ng · h/mL) C_(max) (ng/mL) 84.6 65.368.2 74 83.9 63.3 56.9 95.8 62.1 86 74.0 12.9 17.4 T_(max) (hours) 4 4 25 5 4 5 3 3 4 3.9 1.0 25.5 T_(1/2) (hours) 8.78 9.59 10.02 13.26 9.2410.41 12.8 8.05 10.92 9.47 10.3 1.7 16.3

TABLE 68 Individual subject L-lysine-d-amphetamine intact conjugateconcentrations and pharmacokinetic parameters following oraladministration of a 75 mg dose of L-lysine-d-amphetamine to humansSubject Subject Subject Subject Subject Subject Subject Subject 101 104106 108 109 111 114 115 Subject 118 Subject 119 Mean SD CV % Time Hours0 0 0 0 0 0 0 0 0 0 0 0 0 0   0.5 10.4 22.6 6.92 10.3 0 9.21 7.88 14.587.8 35.5 20.5 25.6 124.7 1 48 40.5 29 41.5 21.2 30.8 23.4 127 88.9 80.153.0 34.6 65.2   1.5 28.4 15.7 16.1 20.3 26.5 19 12.7 38.7 28.6 38 24.49.2 37.5 2 8.87 5.53 4.91 9 18.1 5.62 6.29 12.1 9.75 11.3 9.1 4.0 44.0 32.15 1.29 1.76 1.82 10.6 0 2.31 2.57 1.73 1.73 2.6 2.9 111.6 4 0 0 1.090 4.65 0 1.53 1.01 0 0 0.8 1.5 176.9 5 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 00 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 00 0 10  0 0 0 0 0 0 0 0 0 0 0 0 0 12  0 0 0 0 0 0 0 0 0 0 0 0 0 16  0 00 0 0 0 0 0 0 0 0 0 0 24  0 0 0 0 0 0 0 0 0 0 0 0 0 48  0 0 0 0 0 0 0 00 0 0 0 0 72  0 0 0 0 0 0 0 0 0 0 0 0 0 Parameter AUC_(last) 51.2 44.232.0 43.7 50.4 30.9 29.8 102.1 110.8 86.1 58.1 30.2 52.0 (ng · h/mL)AUC_(inf) 52.5 45.0 33.0 44.9 52.3 34.2 31.4 102.9 111.7 87.0 59.5 29.950.2 (ng · h/mL) C_(max) (ng/mL) 48.0 40.5 29.0 41.5 26.5 30.8 23.4127.0 88.9 80.1 53.6 34.1 63.6 T_(max) (hours) 1 1 1 1 1.5 1 1 1 1 11.05 0.16 15.1 T_(1/2) (hours) 0.43 0.4 0.61 0.43 1.02 0.41 0.75 0.560.38 0.35 0.534 0.211 39.6

TABLE 69 Individual subject d-amphetamine concentrations andpharmacokinetic parameters following oral administration of a 35 mg doseof Adderall XR ® (equivalent to 75 mg dose of L-lysine-d-amphetaminebased on amphetamine base content) to humans Subject Subject SubjectSubject Subject Subject Subject Subject Subject Subject CV 101 104 106108 109 111 114 115 118 119 Mean SD % Time Hours 0 0 0 0 0 0 0 0 0 0 0 00 0   0.5 7.9 2.3 2.8 0.6 2.2 5.7 0 16 2.3 5.3 4.5 4.7 104.3 1 37.6 28.923.3 13.7 29.8 38.2 17.9 46.2 28.8 48.8 31.3 11.5 36.6   1.5 49.9 42.331.1 23.7 39.1 34.4 30.8 65.4 34.1 53 40.4 12.5 31.0 2 65.9 45.8 29.237.4 46.2 65.4 40 64.4 37 67.8 49.9 14.6 29.2 3 95.3 51.7 36.7 23.6 64.762.9 44.7 56.5 31.1 64.8 53.2 20.7 38.9 4 83.7 73.3 56.7 40 67 76.6 56.353.1 33.5 73.3 61.4 16.3 26.6 5 77.4 75.2 71.6 62.1 75.9 76.4 51.5 61.456.8 82.4 69.1 10.3 14.9 6 71.5 72.1 64 59.8 66.9 63.5 56.8 59.8 58.785.7 65.9 8.7 13.2 7 72.3 63.6 71 57.9 70.6 69.7 51.9 48.1 53.7 79.763.9 10.5 16.4 8 60.4 57.1 53.8 53 72 66.9 56.2 56.4 51.7 66.7 59.4 6.911.6 10  50.4 45.5 53 50.7 67.6 57.4 49.1 66.6 48 71.3 56.0 9.3 16.6 12 42.5 41.3 45.4 32.9 53.1 46 37.3 74.7 42.2 60.2 47.6 12.2 25.7 16  31.129.6 35.7 39 45.2 33.9 34.3 64.9 29 40.5 38.3 10.6 27.7 24  14.9 15.122.1 19.5 21.7 21.2 20.7 35.7 17.9 20.5 20.9 5.8 27.7 48  2.5 4.2 3.85.9 5.4 3.8 7.3 5.1 3.9 3 4.5 1.4 32.1 72  0 0.3 1 1 0.3 1.1 2.7 0.3 0 00.7 0.8 124.7 Parameter AUC_(0-12 h) 731.2 625.0 582.6 504.3 711.6 698.5535.4 683.5 509.8 793.2 637.5 101.1 15.9 (ng · h/mL) AUC_(last) 12701230 1343 1269 1568 1436 1354 1920 1101 1520 1401.1 229.0 16.3 (ng ·h/mL) AUC_(inf) 1301 1234 1358 1286 1571 1454 1418 1923 1164 1557 1426.6218.9 15.3 (ng · h/mL) C_(max) (ng/mL) 95.3 75.2 71.5 62 75.9 76.5 56.874.7 58.8 85.8 73.3 11.9 16.3 T_(max) (hours) 3 5 5 5 5 4 6 12 6 6 5.702.41 42.2 T_(1/2) (hours) 8.65 9.01 10.57 11.58 8.37 10.78 16.4 7.2511.05 8.54 10.22 2.59 25.3

TABLE 70 Individual subject d-amphetamine concentrations andpharmacokinetic parameters following oral administration of a 30 mg doseof Dexedrine Spansule ® (equivalent to 75 mg dose ofL-lysine-d-amphetamine based on amphetamine base content) to humansSubject Subject Subject Subject Subject Subject Subject Subject Subject102 103 105 107 110 112 113 116 117 Subject 120 Mean SD CV % Time Hours0 0 0 0 0 0 0 0 0 0 0 0 0 0   0.5 1.2 2.68 1.37 1.4 1.16 2.36 6.75 2.634.95 3.43 2.8 1.8 65.5 1 14.8 26.5 16.7 21.4 25.2 12.7 33.1 22.3 26 21.522.0 6.1 27.8   1.5 24.2 36.9 23.2 28.5 37.2 21.3 42.4 29.2 33.7 39.231.6 7.3 23.2 2 28.6 43.4 27.3 34.6 38.5 27.6 46.2 31.3 38.5 42 35.8 6.919.4 3 27.4 37.3 30.6 40.1 41.7 30.9 52 36.5 42.9 60.1 40.0 10.0 25.2 427.1 44.1 33.5 48.7 45.2 34.7 49.1 40.7 42.4 53.2 41.9 8.1 19.2 5 35.153 40.2 43.4 46.5 42.4 58.1 47 52.1 68.7 48.7 9.7 20.0 6 33.8 58.5 40.246.5 43.5 37.5 56.2 40 51 63 47.0 9.8 20.8 7 37.2 50.7 31.2 41.4 44.9 4257.8 43.6 51.6 65.7 46.6 10.1 21.7 8 35.9 54.3 34.9 45 45 36 58.7 41.853.9 59.2 46.5 9.5 20.4 10  33.1 49.1 34.3 35.5 45 37 51.4 38.9 46.360.1 43.1 8.8 20.4 12  34 51 28.6 34.1 40.8 32.6 51.6 37.7 38.1 50.939.9 8.4 21.1 16  30.2 40.8 25.2 28 33 25.8 41 26.8 29.6 44.9 32.5 7.122.0 24  20.5 27.8 18.2 19.5 17.1 17.8 22.5 19.1 15.5 27.3 20.5 4.2 20.348  3.83 6.89 3.7 5.11 2.56 4.31 6.51 4.43 2.77 5.47 4.6 1.4 31.8 72 0.715 1.63 1 1.7 0 0.622 1.29 1.22 0 1.31 0.9 0.6 64.0 ParameterAUC_(0-12 h) 356.2 539.8 366.4 444.3 480.8 387.0 591.4 436.5 512.8 634.2474.9 94.7 19.9 (ng · h/mL) AUC_(last) 1033 1517 966 1135 1065 1003 14731100 1048 1589 1193 236 19.8 (ng · h/mL) AUC_(inf) 1043 1544 983.5 11681097 1013 1495 1121 1085 1610 1216 238 19.5 (ng · h/mL) C_(max) (ng/mL)37.2 58.5 40.2 48.7 46.5 42.4 58.7 47 53.9 68.7 50.18 9.74 19.4 T_(max)(hours) 7 6 5 4 5 5 8 5 8 5 5.80 1.40 24.1 T_(1/2) (hours) 9.92 11.7412.07 13.8 8.7 10.76 11.47 12.23 9.36 10.92 11.10 1.50 13.6

TABLE 71 Pharmacokinetic parameters of amphetamine following oraladministration of L-lysine-d-amphetamine, Adderall XR ® or DexedrineSpansule ®. Drug L-lysine- L-lysine- d- d- amphetamine amphetamineAdderall Dexedrine Parameter 25 mg Percent¹ 75 mg Percent¹ XR ® Percent¹Spansule ® Percent¹ AUC_(0-12 h) 205.4 33.6   611.5 100  637.5 104  474.9 78 (ng · h/mL) AUC_(last) (ng · h/mL) 396.7 31.5 1237 100 1401.1113 1193 96 AUC_(inf) (ng · h/mL)  415 ± 80.3 32.9 1260 ± 192  100 1429± 223  113 1217 ± 237  97 C_(max) (ng/mL) 25.0 ± 5.57 33.8 74.0 ± 12.9100 73.3 ± 11.9 99 50.2 ± 9.74 68 T_(max) (hours) 3.10 ± 0.88 79.5 3.90± 0.99 100 5.70 ± 2.41 146  5.8 ± 1.40 149 T_(1/2) (hours) 9.66 ± 1.4594 10.3 ± 1.66 100 10.2 ± 2.62 99 11.1 ± 1.48 108 ¹Percent relative toL-lysine-d-amphetamine 75 mg dose

Example 34 Clinical Pharmacokinetic Evaluation and Oral Bioavailabilityof L-lysine-d-amphetamine Dimesylate

In pediatric patients (6-12 yrs) with ADHD, the T_(max) of d-amphetaminewas approximately 3.5 hours following single-dose oral administration ofL-lysine-d-amphetamine dimesylate either 30 mg, 50 mg, or 70 mg after a8-hour overnight fast. See FIG. 65. The T_(max) ofL-lysine-d-amphetamine dimesylate was approximately 1 hour. Linearpharmacokinetics of d-amphetamine after single-dose oral administrationof L-lysine-d-amphetamine dimesylate was established over the dose rangeof 30 mg to 70 mg in children.

TABLE 72 Pharmacokinetic parameters of d-amphetamine andL-lysine-d-amphetamine dimesylate C_(max) T_(max) AUC t_(1/2) Dose(ng/mL) (h) (ng · h/mL) (h) d-amphetamine 30 mg 53.2 ± 9.62 3.41 ± 1.09 845 ± 117 8.90 ± 1.33 50 mg 93.3 ± 18.2 3.58 ± 1.18 1510 ± 242 8.61 ±1.04 70 mg  134 ± 26.1 3.46 ± 1.34 2157 ± 383 8.64 ± 1.32 IntactL-lysine-d-amphetamine dimesylate 30 mg 21.9 0.97 27.9 50 mg 46.0 0.9857.9 70 mg 89.5 1.07 108.9

There is no unexpected accumulation of d-amphetamine at steady state inchildren with ADHD and no accumulation of L-lysine-d-amphetaminedimesylate after once-daily dosing for 7 consecutive days.

Food does not affect the extent of absorption of d-amphetamine inhealthy adults after single-dose oral administration of 70 mg ofL-lysine-d-amphetamine dimesylate capsules but delays T_(max) byapproximately 1 hour (from 3.78 hrs at fasted state to 4.72 hrs after ahigh fat meal). After an 8-hour fast, the extent of absorption ofd-amphetamine following oral administration of L-lysine-d-amphetaminedimesylate in solution and as intact capsules was equivalent.

There were no apparent differences between males and females in exposureas measured by dose-normalized C_(max) and AUC although the range ofvalues in children was higher than that in adults. This is a consequenceof the significant correlation between dose-normalized C_(max) and AUCand body weight and thus the differences are due to the higher doses inmg/kg administered to children. There were no apparent differences int_(1/2) between male and female subjects nor were there any apparentrelationships between t_(1/2) and either age or body weight.

Exemplary results of clinical pharmacokinetic evaluation are presentedin FIG. 66 (AUC), FIG. 67 (C_(max)), and FIG. 68 (T_(max)).

Example 35 Efficacy of L-lysine-d-amphetamine Dimesylate in PediatricClinical Trials

The efficacy of L-lysine-d-amphetamine dimesylate was established in adouble-blind, randomized, placebo-controlled, parallel-group studyconducted in children aged 6-12 (N=290) who met DSM-IV criteria for ADHD(either the combined type or the hyperactive-impulsive type). Patientswere randomized to fixed dose treatment groups receiving final doses of30, 50, or 70 mg of L-lysine-d-amphetamine dimesylate or placebo oncedaily in the morning for four weeks. For patients randomized to 50 and70 mg L-lysine-d-amphetamine dimesylate, dosage was increased by forcedtitration. Significant improvements in the signs and symptoms of ADHD,as rated by investigators (ADHD Rating Scale; ADHD-RS) and parents(Connor's Parent Rating Scale; CPRS), were demonstrated for allL-lysine-d-amphetamine dimesylate doses compared to placebo, for allfour weeks, including the first week of treatment, when allL-lysine-d-amphetamine dimesylate patients were receiving a dose of 30mg/day. Additional dose-responsive improvement was demonstrated in the50 and 70 mg groups, respectively. L-lysine-d-amphetaminedimesylate-treated patients showed significant improvements, as measuredby CPRS scores, in the morning (˜10 am), afternoon (˜2 pm), and evening(˜6 pm) compared with placebo-treated patients, demonstratingeffectiveness throughout the day. The results of the primary efficacyanalysis, ADHD-RS total score change from baseline to endpoint for theITT population, are shown in FIG. 69.

Efficacy was also measured by the SKAMP score. A total of 52 childrenages 6 to 12 who met DSM-IV criteria for ADHD (either the combined typeor the hyperactive-impulsive type) were enrolled in a double-blind,randomized, placebo-controlled crossover study. Patients were randomizedto receive fixed and optimal doses of L-lysine-d-amphetamine (30, 50, 70mg), Adderall XR® (10, 20, or 30 mg), or placebo once daily in themorning for 1 week each treatment. The primary efficacy endpoint in thisstudy was SKAMP-Deportment score (Swanson, Kotkin, Agler, M. Flynn andPelham rating scale). Both L-lysine-d-amphetamine and Adderall XR® werehighly effective compared to placebo. The significant effects ofL-lysine-d-amphetamine occurred within 2 hours post morning dose andcontinued throughout the last assessment time point, 12 hours postmorning dose, compared to placebo, yielding a 12-hour duration ofaction. See FIG. 70.

Example 36 Abuse Liability of Intravenous L-lysine-d-amphetamine

L-lysine-d-amphetamine 50 mg, d-amphetamine 20 mg, and placebo weregiven intravenously over 2 minutes at 48 hour intervals to 9 stimulantabusers in a double blind crossover design to assess abuse liability.Drugs were given according to 3×3 balanced latin squares. Each dosingday, vital sign measures and subjective and behavioral effects wereassessed with questionnaires before dosing and at 0.5, 1, 1.5, 2, 3, 4,5, 6, 9, 12, 16 and 24 hours after dosing. At these times and at 5minutes, a blood sample (5 ml) was taken for d-amphetamine levels.

For d-amphetamine, mean peak plasma level of 77.7 ng/ml of d-amphetamineoccurred at 5 minutes and then rapidly subsided. Administration ofd-amphetamine produced expected d-amphetamine-like effects with meanpeak responses at 15 minutes. The mean maximum response to d-amphetamineon the primary variable of Subject Liking VAS was significantly greaterthan placebo (p=0.01).

For L-lysine-d-amphetamine, mean peak plasma level of 33.8 ng/ml ofd-amphetamine occurred at 3 hours and remained at this level through the4 hour observation. L-lysine-d-amphetamine produced d-amphetamine-likesubjective, behavioral, and vital sign effects with mean peak responsesat 1 to 3 hours. For the primary variable of Subject Liking VAS, theresponse was not greater than placebo (p=0.29). Changes in bloodpressure following L-lysine-d-amphetamine were significant.

At the end of the study, subjects were asked which treatment they wouldtake again. Six subjects chose d-amphetamine 20 mg, two subjects chosenone of the treatments, and one subject chose L-lysine-d-amphetamine 50mg. In summary, L-lysine-d-amphetamine 50 mg did not produce euphoria oramphetamine-like subjective effects although there were late occurringblood pressure increases. The findings suggest thatL-lysine-d-amphetamine itself is inactive. After 1 to 2 hours,L-lysine-d-amphetamine is converted to d-amphetamine. Takenintravenously, L-lysine-d-amphetamine has significantly less abusepotential than immediate release d-amphetamine containing an equalamount of d-amphetamine base.

Example 37 Preliminary Estimates of Decreased Abuse Liability withL-lysine-d-amphetamine vs. d-amphetamine in Healthy Adults with aHistory of Stimulant Abuse

This randomized, single-center, single-blind, dose-escalation study usedpharmacokinetic parameters to obtain preliminary estimates of abuseliability for L-lysine-d-amphetamine (30-150 mg) vs. d-amphetaminesulfate (40 mg) and placebo in healthy adults meeting DSM-IV criteriafor stimulant abuse. Subjects were divided into 3 cohorts of 4 patientseach; all received single doses of L-lysine-d-amphetamine at a minimuminterval of 48 hours, with d-amphetamine sulfate (40 mg) and placeborandomly dispersed. Cohort 1 was administered L-lysine-d-amphetaminedoses of 30, 50, 70, 100 mg; cohort 2 received 50, 70, 100, 130 mgdoses; and cohort 3 received 70, 100, 130, and 150 mg doses.

AUC_(last) d-amphetamine over the first 4 hours was substantially lowerwith 100 mg L-lysine-d-amphetamine (165.3-213.1 ng/mL) vs. 40 mgd-amphetamine (245.5-316.8 ng/mL). C_(max) and AUC_(last) increased withdose for 30-130 mg L-lysine-d-amphetamine, attenuating between the 130mg and 150 mg dose. T_(max) ranged from 3.78-4.25 h withL-lysine-d-amphetamine vs. d-amphetamine sulfate (1.88-2.74 h). Thehalf-life of L-lysine-d-amphetamine (range, 0.44-0.76 h) indicated rapidclearance. Adverse reactions were mild in severity with no significantchanges in vital signs or ECG parameters. L-lysine-d-amphetamine had aslower release of d-amphetamine compared with d-amphetamine sulfate. Atdoses as high as 150 mg, there appears to be an attenuation of themaximum concentration, suggesting higher doses of L-lysine-d-amphetaminewill not lead to further increases in C_(max) and AUC_(last). Theseresults suggest a drug profile consistent with reduced abuse liability.

It will be understood that the specific embodiments of the inventionshown and described herein are exemplary only. Numerous variations,changes, substitutions and equivalents will occur to those skilled inthe art without departing from the spirit and scope of the invention. Inparticular, the terms used in this application should be read broadly inlight of similar terms used in the related applications. Accordingly, itis intended that all subject matter described herein and shown in theaccompanying drawings be regarded as illustrative only and not in alimiting sense and that the scope of the invention be solely determinedby the appended claims.

1. A pharmaceutical composition comprising (a) L-lysine-d-amphetamine ora pharmaceutically acceptable salt thereof and (b) a pharmaceuticallyacceptable additive.
 2. A pharmaceutical composition as defined in claim1, wherein said pharmaceutical composition is suitable for oral dosing.3. A pharmaceutical composition as defined in claim 2, comprising from2.5 mg to 500 mg of L-lysine-d-amphetamine or a pharmaceuticallyacceptable salt thereof.
 4. A pharmaceutical composition as defined inclaim 3, comprising from 10 mg to 250 mg of L-lysine-d-amphetamine or apharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition as defined in claim 4, comprising from 25 mg to 75 mg ofL-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof. 6.A pharmaceutical composition as defined in claim 5, comprising 30 mg ofL-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof. 7.A pharmaceutical composition as defined in claim 5, comprising 50 mg ofL-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof. 8.A pharmaceutical composition as defined in claim 5, comprising 70 mg ofL-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof. 9.A pharmaceutical composition as defined in claim 2, comprising a salt ofL-lysine-d-amphetamine.
 10. A pharmaceutical composition as defined inclaim 9, wherein said salt is a mesylate salt.
 11. A method as definedin claim 10, wherein said salt is a dimesylate salt.
 12. Apharmaceutical composition comprising L-lysine-d-amphetamine dimesylateand a pharmaceutically acceptable additive, wherein said pharmaceuticalcomposition has a water content of about 1% to about 10% by Karl Fischeranalysis.
 13. A pharmaceutical composition as defined in claim 12,wherein said pharmaceutical composition has a water content of about 1%to about 8% by Karl Fischer analysis.
 14. A pharmaceutical compositionas defined in claim 13, wherein said water content is about 2% to about7% by Karl Fischer analysis.